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Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1
ISSN 1064-3745
E-ISBN 978-1-59745-304-2
Library of Congress Control Number: 2007924172](https://image.slidesharecdn.com/1244162-250518072028-1c5ab56e/85/Microarrays-Volume-2-Applications-And-Data-Analysis-Methods-In-Molecular-Biology-V382-2nd-Jang-B-Rampal-6-320.jpg)
![hybridization conditions are repeated with dye assignments reversed in the sec-
ond hybridization in order to minimize systematic bias introduced by differ-
ences in the intensities of the two dyes. A major drawback of this design is that
it becomes increasingly less feasible in terms of cost and materials as more
samples are added to an experiment.
Consequently, for experiments that measure differences in a large number
of samples (e.g., over a developmental time-course) (7,29) or repeated meas-
urements of fluctuating phenomena (27), a reference design, where each
experimental sample is compared with a common reference, is often used. The
indirect expression values obtained from such a reference design have been
shown to be robust when directly compared with those from all-pair experi-
mental design (47). Ideally, a reference sample should represent the genes
expressed in all samples and with intermediate expression levels (7). The
abundance of amphibian and fish embryos that can be obtained and their dis-
crete staging lends itself to the creation of such a pool. Additionally, the use
of a universal reference sample for a given organism facilitates the compari-
son of microarray data sets generated in different experiments, which is oth-
erwise an impractical task. Initial steps to define a common reference pool of
RNA have been taken in Xenopus, where RNA from eight embryonic samples
has been used (7,47). In order to correct for subtle variation between differ-
ent preparations of a reference RNA pool, it is necessary to fluorescently
label each batch and compare the resulting signal intensities in a pairwise
hybridization.
4.2. RNA Isolation and Amplification
Pure RNA preparations are critical to ensure reliable gene expression results.
Partially degraded RNA can bias cDNA labeling toward sequences that lie near
the 3′ terminus and therefore, might distort the relative proportions of various
RNA species when hybridized. To preclude this, many investigators use the
Agilent BioAnalyzer (Agilent Technologies, Palo Alto, CA), an instrument capa-
ble of visualizing picogram amounts of material, to measure RNA quality. Total
RNA isolation techniques can roughly be divided into two categories represented
in approximately equal proportions in the literature: (1) those that use phase sepa-
ration to sequester RNA from proteins and DNA (e.g., Invitrogen’s TRIzol
[Invitrogen, Carlsbad, CA]) or (2) column-based chromatography strategies (e.g.,
Qiagen RNeasy, [Qiagen, Valencia, CA] Ambion MegaClear [Ambion, Austin,
TX]). The use of both techniques, which fractionate RNA on a different physical
basis, in conjunction provides significantly better purification than either method
performed independently (46).
In some experimental situations, it is impossible to isolate the substantial
amounts of total RNA (40–60 μg) needed for a typical labeling reaction. Such
Microarrays in Nonmammalian Vertebrates 7](https://image.slidesharecdn.com/1244162-250518072028-1c5ab56e/85/Microarrays-Volume-2-Applications-And-Data-Analysis-Methods-In-Molecular-Biology-V382-2nd-Jang-B-Rampal-29-320.jpg)
![is the case when working with tissue from embryonic explants, portions of
organs, or other small populations of cells. To obtain the necessary template for
target labeling, several RNA amplification methods have been developed. The
predominant procedure relies on using a bacteriophage RNA polymerase to
transcribe a cDNA template into aRNA (antisense RNA). This transcription is
accomplished by synthesizing single-stranded cDNA from experimental RNA
using primers containing a synthetic SP6, T3, or T7 RNA polymerase promoter.
The final aRNA target has been demonstrated to reflect accurately the size,
complexity, and abundance of mRNAs in the original RNA population (48,49).
Several commercial kits are available that implement this method (e.g.,
MessageAmp II, Ambion; RiboAmp, Arcturus [Sunnyvale, CA]; Superscript
system, Invitrogen; BioArray system, Enzo Life Sciences [Farmingdale, NY]).
4.3. cDNA Labeling
Investigators are again presented with multiple options when preparing a
labeled target solution for hybridization to a microarray. To date, most investiga-
tors have chosen to use the cyanine-based dyes Cy3 and Cy5 (GE Healthcare,
Piscataway, NJ) to label cDNA; however, a range of alternative dyes (e.g., Alexa
Fluor dyes, Invitrogen) are available that are reported to be stronger, more resist-
ant to photobleaching, and less sensitive to pH changes (50). Regardless of flu-
orophore choice, labels can be incorporated into cDNA directly through the
inclusion of dye-conjugated nucleotides or through a coupling reaction between
a reactive dye and amine-substituted nucleotides.
Direct labeling techniques are hampered by the ability of reverse transcriptase
to incorporate bulky dye-conjugated nucleotides, resulting in labeled cDNA of
low fluorescent intensity and a subsequent reduction in assay sensitivity.
Nevertheless, many commercial enzymes have been engineered to improve
incorporation of fluorophores (e.g., SuperScript III, Invitrogen; CyScribe, GE
Healthcare; Omniscript, Qiagen), and many investigators have chosen to utilize
this method as it is cost efficient and requires fewer steps. Indirect labeling relies
on amino-allyl nucleotides (which are incorporated as efficiently as unmodified
nucleotides) that are conjugated to an ester-linked dye molecule following
cDNA synthesis. Indirect labeling generally results in a higher overall level of
cDNA fluorescence by greatly reducing enzymatic bias, though is a more
involved process.
The efficiency and yield of a dye labeling reaction is measured using stan-
dard spectroscopy, where the absorbance of the nucleic acid at 260 nm and the
absorbance of the dye at its emission maximum are measured. Because the vol-
umes involved are generally quite small, these measurements are usually taken
in microcuvettes (10–200 μL) or spectrophotometers specifically designed for
small sample volumes (e.g., the Nanodrop, Nanodrop Technologies).
8 Sipe and Saha](https://image.slidesharecdn.com/1244162-250518072028-1c5ab56e/85/Microarrays-Volume-2-Applications-And-Data-Analysis-Methods-In-Molecular-Biology-V382-2nd-Jang-B-Rampal-30-320.jpg)
![smoothing [LOESS] method), and a final ratio of intensities (experimental/reference
or experimental/control) is calculated for each spot.
4.7.1. Pattern Discovery
Numerous techniques have been developed to aid researchers in discovering
and visualizing patterns present in gene expression data generated by microar-
ray experiments (52). In general, these techniques are excellent choices for ini-
tial data analysis, as they can suggest gene interactions or members of
functional pathways (46). Probably the most prevalent of these computational
methods is cluster analysis, which constructs relationships based on expres-
sion patterns observed in data sets as a whole (53,54). Both treatment groups
and genes can be clustered by some measure of similarity (which vary by
method) to organize samples together into familiar tree-like dendograms.
Those genes (or groups having genes), which are up- or downregulated in a
roughly similar manner lie closest to one another in the tree. Another family of
pattern discovery techniques, which includes principal component analysis
and multidimensional scaling, strives to reduce the number of variables needed
to represent a data set while retaining a maximum amount of its variability
(55). The most informative (i.e., those contributing the most variability to the
dataset as a whole) variables are then projected into two- or three-dimensional
space, and can separate treatments or gene clusters into revealing groups.
4.7.2. Statistical Analysis
The statistical treatment of microarray data has been extensively treated in
the literature (for detailed reviews, see refs. 52,56 and 57). Until relatively
recently, fold changes in gene expression were widely used to select candidate
genes from a data set for further study (as in ref. 13). However, this approach
has been the object of criticism, as it does not give a measure of how signifi-
cant such a difference is likely to be (57). As a result, a number of different sta-
tistical tests have been utilized in the previously listed references to detect
significantly differentially expressed genes in microarray data sets, including
the Students’ t-test (21,29,30,33), F-test (10), analysis of variance (37,38,58),
and Bayesian analyses (16). Another statistical method, Significance analysis of
microarrays, was developed by Tusher (59) specifically for microarrays and has
gained widespread acceptance in the literature (14,25,39).
4.7.3. Independent Confirmation of Results
It is important to note that the data obtained from a single microarray exper-
iment will only implicate genes as candidates for involvement in a given net-
work or pathway. It is up to the investigator to decide whether the results are
accurate for the particular biological system under study (60). Consequently,
Microarrays in Nonmammalian Vertebrates 11](https://image.slidesharecdn.com/1244162-250518072028-1c5ab56e/85/Microarrays-Volume-2-Applications-And-Data-Analysis-Methods-In-Molecular-Biology-V382-2nd-Jang-B-Rampal-33-320.jpg)
![The cost of the slides might increase from simple silane slides to linker-
modified and then more from polymer to dendrimer slides. The following para-
graphs provide recommendations on the selection of suitable surfaces for various
applications (1).
2.2.2. Oligonucleotide Microarrays
Now-a-days oligonucleotide arrays can be considered a standard application
featuring a number of advantages over cDNA arrays. Because oligonucleotides
modified with a nucleophilic linker react under mild conditions with elec-
trophilic groups including glycidyloxy (epoxy), aldehyde, isothiocyanate, and
activated ester (e.g., N-hydroxysuccinimide [NHS]), slides presenting these
functional groups are preferably used. Aminosilane-coated slides does not pro-
vide a suitable functionality to covalently immobilize oligonucleotides in a mild
and specific manner. On these slides, immobilization can only be achieved by
applying harsh conditions, such as heating to 80°C or UV crosslinking (2). In
many articles, epoxysilane- and aldehyde-modified slides (prepared by the
reaction of aminosilane slides with glutaraldehyde) are successfully used for
oligonucleotide applications (refer to publications). However, when comparing
processing protocols for epoxysilane- and aldehyde-modified slides, the former
has clear advantage regarding simplicity, processing time, flexibility with
regard to the blocking reagent, no harmful and toxic chemicals (sodium boro-
hydride and sensitive) and hence less cost for waste disposal. It has been found
that there is no need for a (rather expensive and sensitive) nucleophilic linker
group to immobilize long oligonucleotides at surfaces presenting epoxy
groups (see Chapter 3).
22 Sobek et al.
Fig. 3. Schematic representation of slide surface architecture. (A) Simple silane-
based slides. (B) Linker-modified slides. (C) Polymer-coated slides. (D) Dendrimer-
coated slides. The representation is simplified such that no structural effects are taken
into account (molecular orientational effects, silane multilayer formation, cross-linking
of polymers, and gel formation). Black spheres are linking groups introduced by the
chemical synthesis of the slide. Gray spheres are reactive groups. On polymer slides of
type 3C these groups might be either reactive functional groups or nonreactive groups,
for example, amide. Structures are not drawn to scale.](https://image.slidesharecdn.com/1244162-250518072028-1c5ab56e/85/Microarrays-Volume-2-Applications-And-Data-Analysis-Methods-In-Molecular-Biology-V382-2nd-Jang-B-Rampal-44-320.jpg)
![This ebook is for the use of anyone anywhere in the United
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eBook.
Title: Annali d'Italia, vol. 2
Author: Lodovico Antonio Muratori
Release date: August 27, 2013 [eBook #43575]
Most recently updated: October 23, 2024
Language: Italian
Credits: Produced by Carlo Traverso, Claudio Paganelli, Barbara
Magni and the Online Distributed Proofreading Team at
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*** START OF THE PROJECT GUTENBERG EBOOK ANNALI D'ITALIA,
VOL. 2 ***](https://image.slidesharecdn.com/1244162-250518072028-1c5ab56e/85/Microarrays-Volume-2-Applications-And-Data-Analysis-Methods-In-Molecular-Biology-V382-2nd-Jang-B-Rampal-57-320.jpg)
![Anno di
Cristo CCCXLI. Indizione XIV.
Giulio papa 5.
Costanzo e
Costante imperadori 5.
Consoli
Antonio Marcellino e Petronio Probino.
Un'iscrizione che si legge nella mia Raccolta [Thes. Novus Inscript.,
pag. 377.], quando pur sia indubitata reliquia dell'antichità, ci assicura
dei nomi di questi consoli, in addietro ignoti. Aurelio Celsino dal dì 25
di febbraio cominciò ad esercitare la prefettura di Roma. Sul fine di
giugno diede Costanzo Augusto una legge in Lauriaco [L. 31, de
Decurion., Cod. Theodos.], creduto dal Gotofredo luogo della Batavia, ma
che più verisimilmente fu il Lauriaco, luogo insigne e colonia de'
Romani, posta alle parti superiori del Danubio. Era questo principe
divenuto signor delle Gallie, e colà dovette accorrere [Idacius, in
Fastis.], perchè i Franchi, passato il Reno, metteano a sacco le vicine
contrade romane. Abbiamo da san Girolamo [Hieron., in Chron.] che
seguirono fra que' Barbari e le armate di Costante varii
combattimenti, ma senza dichiararsi la fortuna per alcuna delle parti.
Libanio [Liban., Orat. III.], descrivendo a lungo i costumi e il genio de'
Franchi d'allora, li dipinge per gente turbolenta ed inquieta, a cui il](https://image.slidesharecdn.com/1244162-250518072028-1c5ab56e/85/Microarrays-Volume-2-Applications-And-Data-Analysis-Methods-In-Molecular-Biology-V382-2nd-Jang-B-Rampal-63-320.jpg)
![riposo riusciva un supplizio. Solamente nell'anno seguente ebbe fine
questa guerra. Tanto il medesimo san Girolamo che Idacio mettono
sotto il presente anno spaventosi tremuoti che fecero traballare
moltissime città dell'Oriente. Tennero in quest'anno gli ariani un
conciliabolo in Antiochia, per alterare i decreti sacrosanti del concilio
niceno. Appena terminata fu la sacrilega loro assemblea, che il
tremuoto cominciò a scuotere orribilmente la misera città, siccome
attestano Socrate [Socrates, Histor., lib. 2, cap. 11.] e Sozomeno
[Sozomenus, Histor., lib. 3, cap. 6.], e quasi per un anno si andarono
sentendo varie altre scosse. Non parla Teofane [Theophanes, in
Chronogr.] se non di tre giorni, ne' quali probabilmente quella città fu
in maggior pericolo. Lo stesso autore nota che circa questi tempi
Costanzo Augusto cinse di forti mura e fortificò in altre guise Amida,
città della Mesopotamia, situata presso il fiume Tigri, acciocchè
servisse di antemurale contro ai Persiani. Ammiano [Ammianus, Histor.,
lib. 18, cap. 9.], scrittore di maggior credito, all'incontro, scrive che
molto prima d'ora, cioè vivente ancora il padre, Costanzo Cesare con
torri e mura fece divenir quel luogo un'importante fortezza, di cui
sempre più crebbe la popolazione e la fama ne' tempi susseguenti.
Durava tuttavia la guerra coi Persiani, ovvero, se Socrate [Socrat.,
Histor., lib. 2, cap. 25.] non s'inganna, essa ebbe principio in questi
medesimi tempi; ma quali azioni militari si facessero, non è
pervenuto a nostra notizia. Già abbiam detto che Costantino il
Grande con varii editti e in altre guise si studiò di abolir le
superstizioni del paganesimo, distrusse moltissimi templi de' gentili,
vietò gli empii loro sagrifizii: il che vien confermato da Socrate [Idem,
ibid., lib. 1, cap. 8.], da Teodoreto [Theodoret., in Histor. Eccl.], da Teofane
[Theoph., Chronogr.] e da altri. Ma lo svellere dal cuore di tanta gente
gli antichi errori e riti, difficil cosa riusciva nella pratica. Costante
Augusto nell'anno presente, siccome principe di massime cattoliche e
di zelo cristiano, per eseguire eziandio ciò che il padre gli avea
premurosamente raccomandato, pubblicò una legge, con cui,
confermando gli editti paterni [L. 2, de Paganis., Cod. Theod.], sotto
rigorose pene abolisce i sagrifizii de' pagani, e per conseguenza
ancora il culto degl'idoli. Siffatti editti, e l'esempio de' principi seguaci
della legge di Cristo, furono quegli arieti che diedero un gran tracollo](https://image.slidesharecdn.com/1244162-250518072028-1c5ab56e/85/Microarrays-Volume-2-Applications-And-Data-Analysis-Methods-In-Molecular-Biology-V382-2nd-Jang-B-Rampal-64-320.jpg)
![al gentilesimo, con ridurlo a poco a poco all'ultima rovina. Ma se ad
occhio veniva meno la falsa religion de' pagani, per cura
massimamente dell'Augusto Costante, andavano ben crescendo in
questi tempi le forze dell'arianismo in Oriente con discapito della
Chiesa cattolica, per la protezion che avea preso di quella fazione
l'Augusto Costanzo. Le insigni sedie episcopali di Alessandria,
Antiochia e Costantinopoli vennero in questi tempi occupate da'
vescovi ariani [Socrat., lib. 5, cap. 7. Theoph. Cedr.]: e tutte le chiese
d'essa città di Costantinopoli caddero in poter de' medesimi eretici.
Ma intorno a ciò è da consultare la storia ecclesiastica. Grande
solennità nel presente anno fu fatta in Antiochia per la dedicazione di
questa magnifica cattedrale, cominciata da Costantino il Grande, e
compiuta solamente ora per cura del suddetto imperadore Costanzo.](https://image.slidesharecdn.com/1244162-250518072028-1c5ab56e/85/Microarrays-Volume-2-Applications-And-Data-Analysis-Methods-In-Molecular-Biology-V382-2nd-Jang-B-Rampal-65-320.jpg)
![Anno di
Cristo CCCXLII. Indizione XV.
Giulio papa 6.
Costanzo e
Costante imperadori 6.
Consoli
Flavio Giulio Costanzo Augusto per la terza volta e Flavio Giulio
Costante Augusto per la seconda.
Ad Aurelio Celsino nella prefettura di Roma succedette in
quest'anno nelle calende d'aprile Mavorzio Lolliano [ Cuspinianus.
Panvinius. Bucherius.], il cui impiego durò sino al dì 14 di luglio, con
avere per successore Acone (ossia Aconio) Catulino (ossia Catullino)
Filomazio (o pur Filoniano). All'anno presente riferisce il Gotofredo [
Gothofred., Chron. Cod. Theodos.] un editto [ L. 3, de Paganis, Cod. eod.
Theod.] di Costante Augusto, dato nel dì primo di novembre, e
indirizzato al medesimo Catullino prefetto di Roma, in cui ordina che,
quantunque s'abbia da abolire affatto la superstizione pagana, pure
non si demoliscano i templi situati fuori di Roma, per non levare al
popolo romano i divertimenti dei giuochi circensi e combattimenti
che aveano presa la origine da que' medesimi templi. Nè già paresse
per questo raffreddato punto lo zelo di questo principe in favore del
cristianesimo, perchè egli non altro volle che conservar le mura e le](https://image.slidesharecdn.com/1244162-250518072028-1c5ab56e/85/Microarrays-Volume-2-Applications-And-Data-Analysis-Methods-In-Molecular-Biology-V382-2nd-Jang-B-Rampal-66-320.jpg)
![fabbriche materiali di que' templi, ma con obbligo di sbarbicar tutto
quel che sapeva di superstizione gentilesca, come idoli, altari e
sagrifizii. Fors'anche non dispiaceva ad alcuni accorti cristiani che
restassero in piedi que' superbi edifizii, per convertirli un dì in onore
del vero Dio. Ma che in tanti altri luoghi venissero abbattuti i templi
de' gentili, Giulio Firmico [Julius Firmicus, de error. prof. Rel.], che circa
questi tempi fioriva e scrisse i suoi libri, ce ne assicura. Fino al
presente anno sostennero i Franchi la guerra nelle Gallie contra
dell'Augusto Costante [ Hieronymus, in Chron. Idacius, in Fastis. Socrates, lib.
2, cap. 13. Theoph., in Chron.]. Tali percosse nondimeno dovettero
riportare dall'armi romane, che finalmente si ridussero a chiedere
pace. Un trattato di amicizia e lega conchiuso con Costante li fece
ripassare il Reno. Libanio [ Liban., Orat. III.] con oratoria
magniloquenza lasciò scritto che il solo terrore del nome di Costante
obbligò que' popoli barbari ad implorare un accordo, senza dire che
fossero domati coll'armi, come scrissero tanti altri. Aggiugne ch'essi
Franchi riceverono dalla mano di Costante i loro principi, e stettero
poi quieti per qualche tempo. Occorse nell'anno presente in
Costantinopoli più d'una sedizione fra i cattolici ed ariani [ Socrates, lib.
2, cap. 13. Sozomenus, Hist. Eccl. Idacius, in Fastis. Hieronym., in Chron.], da
che Costanzo Augusto, sposata affatto la fazione degli ultimi, mandò
ordine che fosse da quella cattedra cacciato Paolo vescovo cattolico,
per introdurvi Macedonio ariano. Crebbe un dì a tal segno
l'impazienza e il furor della plebe cattolica, che andarono ad
incendiar la casa di Ermogene generale dell'armi, a cui era venuto
l'ordine dell'imperadore di eseguir la deposizione del vescovo
cattolico; e messe le mani addosso al medesimo Ermogene, lo
strascinarono per la città, e lo uccisero. Costanzo, che allora si
trovava ad Antiochia, udita cotal novità, tosto per le poste volò a
Costantinopoli: cacciò Paolo e gastigò il popolo, con privarlo della
metà del grano, che per istituzione di Costantino gli era
somministrato gratis ogni anno; cioè di ottanta mila moggia o misure
ridusse il dono a sole quaranta mila.](https://image.slidesharecdn.com/1244162-250518072028-1c5ab56e/85/Microarrays-Volume-2-Applications-And-Data-Analysis-Methods-In-Molecular-Biology-V382-2nd-Jang-B-Rampal-67-320.jpg)
![Anno di
Cristo CCCXLIII. Indizione I.
Giulio papa 7.
Costanzo e
Costante imperadori 7.
Consoli
Marco Mecio Memmio Furio Baburio Ceciliano Procolo e Romolo.
Questa gran filza di cognomi data al primo console, cioè a
Procolo, si truova in un'iscrizione creduta spettante a lui, e
rapportata dal Panvinio e Grutero. Non Balburio, come essi hanno,
ma Baburio viene appellato nelle schede di Ciriaco, che riferisce lo
stesso marmo. Il secondo console dal suddetto Panvinio, che cita
un'iscrizione, vien chiamato Flavio Pisidio Romolo. Vopisco, nella Vita
d'Aureliano [Vopiscus, in Aurel.], ci rappresenta questo Procolo per
uomo abbondante, non so se più di ricchezze o di vanità, scrivendo
essersi poco fa veduto il consolato di Furio Procolo solennizzato con
tale sfoggio nel circo, che non già premii, ma patrimonii interi parve
che fossero donati ai vincitori nella corsa de' cavalli. Ci fan conoscere
tali parole in che tempo Vopisco fiorisse e scrivesse. Nella prefettura
di Roma continuò ancora per quest'anno Aconio Catullino. Dappoichè
la pace stabilita coi Franchi rimise la calma in tutte le Gallie,
Costante Augusto, il quale si truovava in Bologna di Picardia nel](https://image.slidesharecdn.com/1244162-250518072028-1c5ab56e/85/Microarrays-Volume-2-Applications-And-Data-Analysis-Methods-In-Molecular-Biology-V382-2nd-Jang-B-Rampal-68-320.jpg)
![gennaio dell'anno presente [Gothofred., Chron. Cod. Theodos.], volle farsi
vedere anche ai popoli della Bretagna, e passò nel furore del verno
colà con tutta felicità. Se prestiam fede a Libanio [Liban., Orat. III.],
guerra non v'era che il chiamasse di là dal mare, ma solo timor di
guerra; e da Ammiano Marcellino [Ammianus, lib. 20, cap. 1.] si ha
abbastanza per credere che i Barbari di quella grand'isola avessero
fatta almen qualche scorreria nel paese de' Romani. Per altro, che
non succedessero battaglie e vittorie in quelle parti, si può
argomentare dal suddetto Libanio, giacchè egli di niuna fa menzione.
Truovansi nulladimeno alcune medaglie, dove egli è appellato
[Mediob., in Numismat. Imperator.] debellatore e trionfatore delle nazioni
barbare, le quali, se non sono parti della sola bugiarda adulazione,
possono indicare qualche vantaggio delle sue armi in quelle contrade
ancora. Oltre di che, Giulio Firmico [Julius Firmicus, de error. profan. Rel.],
parlando ai due Augusti, dice, che dopo aver essi abbattuti i templi
de' gentili nell'anno 341, Dio avea prosperate le lor armi; che aveano
vinti i nemici, dilatato l'imperio; che i Britanni, all'improvviso
comparir dell'imperadore, s'erano intimoriti. Truovasi poi esso
Augusto nel dì 30 di giugno ritornato a Treveri, dove è data una sua
legge. Ci fanno poi altre leggi vedere Costanzo Augusto in Antiochia,
in Cizico, in Jerapoli, tutte città dell'Asia, imperocchè non gli lasciava
godere riposo la guerra sempre viva coi Persiani. Osserviamo ancora
in una delle sue leggi [L. 35, de Decur., Cod. Theod.] ch'egli chiamò a
militare in quest'anno i figliuoli dei veterani, purchè giunti all'età di
sedici anni, per bisogno certamente di quella guerra. Non so io dire
quale credenza si meriti Teofane [Theoph., in Chronogr.], allorchè scrive
che circa questi tempi Costanzo, dopo aver vinti gli Assirii, cioè i
Persiani suddetti, trionfò. Niuno de' più antichi e vicini storici a lui
attribuisce alcuna memorabil vittoria di que' popoli, e molto meno un
vero trionfo. Abbiamo inoltre dal medesimo Teofane che la città di
Salamina nell'isola di Cipri per un fierissimo tremuoto restò la
maggior parte smantellata; siccome ancora circa questi tempi ebbe
principio la persecuzione mossa da Sapore re di Persia contra de'
cristiani abitanti ne' paesi di suo dominio.](https://image.slidesharecdn.com/1244162-250518072028-1c5ab56e/85/Microarrays-Volume-2-Applications-And-Data-Analysis-Methods-In-Molecular-Biology-V382-2nd-Jang-B-Rampal-69-320.jpg)
![Anno di
Cristo CCCXLIV. Indizione II.
Giulio papa 8.
Costanzo e
Costante imperadori 8.
Consoli
Leonzio e Sallustio.
Nel dì 11 d'aprile ad Acone, ossia Aconio, Catullino succedette
nella prefettura di Roma Quinto Rustico. Nulla di considerabile ci
somministra per questo anno la storia, se non che truoviamo una
legge [L. 3, de excusat. artific.], con cui Costanzo Augusto concede delle
esenzioni ai professori di meccanica, architettura e ai livellatori delle
acque. Il genio edificatorio veramente non mancò a questo
imperadore, ed egli lasciò molte suntuose fabbriche da lui fatte in
Costantinopoli, Antiochia ed altri luoghi. Ma se egli coll'una mano
innalzava materiali edifizii nel suo dominio, coll'altra incautamente si
studiava di atterrare e distruggere la dottrina e Chiesa cattolica,
lasciandosi aggirare a lor talento dai seguaci dello eresiarca Ario.
Però in questi tempi smisuratamente prevalse in Oriente la lor
fazione: laddove Costante Augusto in Occidente, con dichiararsi
protettore dei dogmi del concilio niceno, divenne scudo della Chiesa
cattolica. Se in Oriente si tenevano conciliaboli contra la fede nicena,](https://image.slidesharecdn.com/1244162-250518072028-1c5ab56e/85/Microarrays-Volume-2-Applications-And-Data-Analysis-Methods-In-Molecular-Biology-V382-2nd-Jang-B-Rampal-70-320.jpg)
![in Occidente ancora si formavano concilii per sostenerla. Ma intorno
a ciò mi rimetto alla storia ecclesiastica. Intanto era flagellato da Dio
l'imperador Costanzo col tarlo della guerra persiana; e benchè
Teofane [Theoph., in Chronogr.] ancora sotto quest'anno racconti che
vennero alle mani le due armate romana e persiana, e che gran
numero di que' Barbari lasciò la vita sul campo; pure, poco o nulla
servirono questi pretesi vantaggi, perchè più che mai vigorosi i
Persiani continuarono a fare il ballo sulle terre romane, senza che
mai riuscisse ai Romani di cavalcare sul paese nemico. Abbiamo poi
da san Girolamo [Hieronymus, in Chronico.] e dal suddetto Teofane che
nell'anno presente Neocesarea, città la più riguardevol del Ponto, fu
interamente rovesciata a terra da un orrendo tremuoto colla morte
della maggior parte del popolo, essendosi solamente salvata la
cattedrale fabbricata da san Gregorio Taumaturgo colla casa
episcopale, dove esso vescovo e chiunque ivi si trovò rimasero esenti
da quello eccidio.](https://image.slidesharecdn.com/1244162-250518072028-1c5ab56e/85/Microarrays-Volume-2-Applications-And-Data-Analysis-Methods-In-Molecular-Biology-V382-2nd-Jang-B-Rampal-71-320.jpg)
![Anno di
Cristo CCCXLV. Indizione III.
Giulio papa 9.
Costanzo e
Costante imperadori 9.
Consoli
Amanzio ed Albino.
Secondo il Catalogo del Cuspiniano e del Bucherio, nel dì 5 di
luglio Probino fu creato prefetto di Roma. Una legge [L. 7, de petition.,
Cod. Theod.] di Costante Augusto, data nel dì 15 maggio, ci fa vedere
questo imperador ritornato dalla Bretagna a Treveri. Però non so se
sussista l'aver creduto il Tillemont [Tillemont, Mémoires des Empereurs et
de l'Histoire Ecclesiastique.] ch'esso Augusto verso il fine del medesimo
mese fosse in Milano, dove invitò lo sbattuto santo Atanasio, per
patrocinarlo contra la prepotenza degli ariani. Certamente cominciò
verso questi tempi il cattolico Augusto a tempestar con lettere il
fratello Costanzo, acciocchè si tenesse un concilio valevole a metter
fine a tante turbolenze della Chiesa. Ma non si arrivò a questo se
non nell'anno 347, siccome allora accenneremo. Da una legge del
Codice Teodosiano [L. 5, de exactionib., Cod. Theod.] apprendiamo che
l'Augusto Costanzo, nel dì 12 di maggio del presente anno, si trovava
in Nisibi città della Mesopotamia, e, senza fallo, per accudire alla](https://image.slidesharecdn.com/1244162-250518072028-1c5ab56e/85/Microarrays-Volume-2-Applications-And-Data-Analysis-Methods-In-Molecular-Biology-V382-2nd-Jang-B-Rampal-72-320.jpg)
![guerra coi Persiani. Abbiamo poi da san Girolamo [Hieron., in Chronico.]
e da Teofane [Theoph., in Chronogr.] che in quest'anno ancora i tremuoti
cagionarono nuove rovine in varie città. Fra le altre la marittima di
Epidamno ossia di Durazzo, città della Dalmazia, restò quasi affatto
abissata. Anche in Roma per tre giorni sì gagliarde furono le scosse,
che si paventò l'universal caduta delle fabbriche. Nella Campania
dodici città andarono per terra; e l'isola, o, vogliam dire, la città di
Rodi, fieramente anch'essa risentì la medesima sciagura. Se
crediamo alla Cronica Alessandrina [Chronic. Alexandrinum.], Costanzo
Augusto cominciò in quest'anno la fabbrica delle sue terme in
Costantinopoli; ma intorno a ciò è da vedere il Du-Cange [Du-Cange,
Hist. Byz.], che rapporta altre notizie spettanti a quell'insigne edifizio.](https://image.slidesharecdn.com/1244162-250518072028-1c5ab56e/85/Microarrays-Volume-2-Applications-And-Data-Analysis-Methods-In-Molecular-Biology-V382-2nd-Jang-B-Rampal-73-320.jpg)
![Anno di
Cristo CCCXLVI. Indizione IV.
Giulio papa 10.
Costanzo e
Costante imperadori 10.
Consoli
Flavio Giulio Costanzo Augusto per la quarta volta e Flavio
Giulio Costante Augusto per la terza.
Perchè non si dovettero speditamente accordare i due Augusti
intorno al prendere insieme il consolato, o pure a notificarlo, noi
troviamo che nel Catalogo del Bucherio e in un concilio di Colonia
per li primi mesi dell'anno presente non si contavano i consoli nuovi;
perciò l'anno veniva indicato colla formola di dopo il consolato di
Amanzio ed Albino. Nella prefettura di Roma stette Probino sino al dì
26 di dicembre dell'anno presente [Cuspinianus. Panvinius. Bucherius.], ed
allora in quella carica succedette Placido. Noi ricaviamo dalle leggi
del Codice Teodosiano [Gothofred., Chronolog. Cod. Theodos.] spettanti a
quest'anno che Costante Augusto era in Cesena nel dì 23 di maggio,
e in Milano nel dì 21 di giugno. Dall'Italia dovette egli passare in
Macedonia, perchè abbiamo una legge di lui data in Tessalonica nel
dì 6 di dicembre. Per conto dell'Augusto Costanzo, egli non altrove
comparisce che in Costantinopoli, dove confermò o pur concedette](https://image.slidesharecdn.com/1244162-250518072028-1c5ab56e/85/Microarrays-Volume-2-Applications-And-Data-Analysis-Methods-In-Molecular-Biology-V382-2nd-Jang-B-Rampal-74-320.jpg)
![molte esenzioni agli ecclesiastici. All'anno presente riferisce san
Girolamo [Hieron., in Chron.] la fabbrica del porto di Seleucia, città
famosa della Soria, poche miglia distante da Antiochia, capitale
dell'Oriente. Anche Giuliano [Julian., Orat. I.] e Libanio [Liban., Orat. III.]
parlano di questa impresa, che riuscì d'incredibile spesa al pubblico,
perchè per formare quel porto non già alla sboccatura del fiume
Oronte, come talun suppone, ma bensì alla stessa Seleucia,
convenne tagliar molti scogli e un pezzo di montagna, che
impedivano l'accesso alle navi, e rendevano pericolosa e poco utile
una specie di porto che quivi anche antecedentemente era. Perchè la
corte dell'imperador Costanzo per lo più soggiornava in Antiochia, di
incredibil comodo e ricchezza riuscì dipoi a quella città il vicino porto
di Seleucia. Teofane [Theophanes, Chronogr.] aggiugne che Costanzo
con altre fabbriche ampliò ed adornò la stessa città di Seleucia; ed
inoltre abbellì la città di Antarado nella Fenicia, la quale prese allora
il nome di Costanza. Mentre poi esso Augusto Costanzo impiegava in
questa maniera i suoi pensieri e i tesori, cavati dalle viscere dei
sudditi, dietro alle fabbriche, il re di Persia Sapore non lasciava in
ozio la forza delle sue armi; e però, secondochè scrive il suddetto
Teofane, nell'anno presente si portò per la seconda volta all'assedio
della città di Nisibi nella Mesopotamia. Vi stette sotto settantotto
giorni, e, non ostante tutti i suoi sforzi, fu in fine obbligato a
vergognosamente levare il campo e ritirarsi. Nella Cronica di san
Girolamo un tale assedio vien riferito all'anno seguente. Ma cotanto
hanno gli antichi moltiplicato il numero degli assedii di Nisibi con
discordia fra loro, che non si sa che credere. Verisimilmente un solo
assedio fin qui fu fatto, cioè se sussiste il già accennato all'anno 338,
un altro non sarà da aggiugnere all'anno presente. Parleremo,
andando innanzi, d'altri assedii di quella città. Pare che in quest'anno
accadesse una sedizione in Costantinopoli, per cui quel governatore
Alessandro restò ferito, e se ne fuggì ad Eraclea. Tornossene ben egli
fra poco al suo impiego, ma poco stette ad esser deposto da
Costanzo, con succedergli in quel governo Limenio. Libanio [Liban., in
ejus vita.] quegli è che ci ha conservata questa notizia, e che sparla
forte d'esso Limenio, perchè il buon sofista fu cacciato da
Costantinopoli d'ordine suo.](https://image.slidesharecdn.com/1244162-250518072028-1c5ab56e/85/Microarrays-Volume-2-Applications-And-Data-Analysis-Methods-In-Molecular-Biology-V382-2nd-Jang-B-Rampal-75-320.jpg)
Microarrays Volume 2 Applications And Data Analysis Methods In Molecular Biology V382 2nd Jang B Rampal
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- 4. M E T H O D S I N M O L E C U L A R B I O L O G Y™ John M. Walker, SERIES EDITOR 383. Cancer Genomics and Proteomics: Methods and Protocols, edited by Paul B. Fisher, 2007 382. Microarrays, Second Edition: Volume 2, Applications and Data Analysis, edited by Jang B. Rampal, 2007 381. Microarrays, Second Edition: Volume 1, Synthesis Methods, edited by Jang B. Rampal, 2007 380. Immunological Tolerance: Methods and Protocols, edited by Paul J. Fairchild, 2007 379. Glycovirology Protocols, edited by Richard J. Sugrue, 2007 378. Monoclonal Antibodies: Methods and Protocols, edited by Maher Albitar, 2007 377. Microarray Data Analysis: Methods and Applications, edited by Michael J. Korenberg, 2007 376. Linkage Disequilibrium and Association Mapping: Analysis and Application, edited by Andrew R. Collins, 2007 375. In Vitro Transcription and Translation Protocols: Second Edition, edited by Guido Grandi, 2007 374. Quantum Dots: Methods and Protocols, edited by Charles Z. Hotz and Marcel Bruchez, 2007 373. Pyrosequencing® Protocols, edited by Sharon Marsh, 2007 372. Mitochondrial Genomics and Proteomics Protocols, edited by Dario Leister and Johannes Herrmann, 2007 371. Biological Aging: Methods and Protocols, edited by Trygve O. Tollefsbol, 2007 370. Adhesion Protein Protocols, Second Edition, edited by Amanda S. Coutts, 2007 369. Electron Microscopy: Methods and Protocols, Second Edition, edited by John Kuo, 2007 368. Cryopreservation and Freeze-Drying Protocols, Second Edition, edited by John G. Day and Glyn Stacey, 2007 367. Mass Spectrometry Data Analysis in Proteomics, edited by Rune Matthiesen, 2007 366. Cardiac Gene Expression: Methods and Protocols, edited by Jun Zhang and Gregg Rokosh, 2007 365. Protein Phosphatase Protocols: edited by Greg Moorhead, 2007 364. Macromolecular Crystallography Protocols: Volume 2, Structure Determination, edited by Sylvie Doublié, 2007 363. Macromolecular Crystallography Protocols: Volume 1, Preparation and Crystallization of Macromolecules, edited by Sylvie Doublié, 2007 362. Circadian Rhythms: Methods and Protocols, edited by Ezio Rosato, 2007 361. Target Discovery and Validation Reviews and Protocols: Emerging Molecular Targets and Treatment Options, Volume 2, edited by Mouldy Sioud, 2007 360. Target Discovery and Validation Reviews and Protocols: Emerging Strategies for Targets and Biomarker Discovery, Volume 1, edited by Mouldy Sioud, 2007 359. Quantitative Proteomics by Mass Spectrom- etry, edited by Salvatore Sechi, 2007 358. Metabolomics: Methods and Protocols, edited by Wolfram Weckwerth, 2007 357. Cardiovascular Proteomics: Methods and Protocols, edited by Fernando Vivanco, 2006 356. High Content Screening: A Powerful Approach to Systems Cell Biology and Drug Discovery, edited by D. Lansing Taylor, Jeffrey Haskins, and Ken Guiliano, 2007 355. Plant Proteomics: Methods and Protocols, edited by Hervé Thiellement, Michel Zivy, Catherine Damerval, and Valerie Mechin, 2006 354. Plant–Pathogen Interactions: Methods and Protocols, edited by Pamela C. Ronald, 2006 353. DNA Analysis by Nonradioactive Probes: Methods and Protocols, edited by Elena Hilario and John. F. MacKay, 2006 352. Protein Engineering Protocols, edited by Kristian Müller and Katja Arndt, 2006 351. C. elegans: Methods and Applications, edited by Kevin Strange, 2006 350. Protein Folding Protocols, edited by Yawen Bai and Ruth Nussinov 2007 349. YAC Protocols, Second Edition, edited by Alasdair MacKenzie, 2006 348. Nuclear Transfer Protocols: Cell Reprogramming and Transgenesis, edited by Paul J. Verma and Alan Trounson, 2006 347. Glycobiology Protocols, edited by Inka Brockhausen-Schutzbach, 2006 346. Dictyostelium discoideum Protocols, edited by Ludwig Eichinger and Francisco Rivero, 2006 345. Diagnostic Bacteriology Protocols, Second Edi- tion, edited by Louise O'Connor, 2006 344. Agrobacterium Protocols, Second Edition: Volume 2, edited by Kan Wang, 2006 343. Agrobacterium Protocols, Second Edition: Volume 1, edited by Kan Wang, 2006 342. MicroRNA Protocols, edited by Shao-Yao Ying, 2006 341. Cell–Cell Interactions: Methods and Protocols, edited by Sean P. Colgan, 2006 340. Protein Design: Methods and Applications, edited by Raphael Guerois and Manuela López de la Paz, 2006 339. Microchip Capillary Electrophoresis: Methods and Protocols, edited by Charles S. Henry, 2006 338. Gene Mapping, Discovery, and Expression: Methods and Protocols, edited by M. Bina, 2006 337. Ion Channels: Methods and Protocols, edited by James D. Stockand and Mark S. Shapiro, 2006
- 5. M E T H O D S I N M O L E C U L A R B I O L O G Y™ Microarrays Volume 2: Applications and Data Analysis SECOND EDITION Edited by Jang B. Rampal Beckman Coulter, Inc. Brea, CA
- 6. © 2007 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 www.humanapress.com All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. Methods in Molecular BiologyTM is a trademark of The Humana- Press Inc. All papers, comments, opinions, conclusions, or recommendations are those of the author(s), and do not necessarily reflect the views of the publisher. This publication is printed on acid-free paper. h ANSI Z39.48-1984 (American Standards Institute) Permanence of Paper for Printed Library Materials. Production Editor: Jennifer Hackworth Cover design by Karen Schulz Cover illustration: Fig. 2, Chapter 4; see complete caption and discussion on p. 63. For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contact Humana at the above address or at any of the following numbers: Tel.: 973-256-1699; Fax: 973-256-8341; E-mail: orders@humanapr.com; or visit our Website: www.humanapress.com Photocopy Authorization Policy: Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Humana Press Inc., provided that the base fee of US $30.00 per copy is paid directly to the Copyright Clearance Center at 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have been granted a photocopy license from the CCC, a separate system of payment has been arranged and is acceptable to Humana Press Inc. The fee code for users of the Transactional Reporting Service is: [978-1-58829-944-4/07 $30.00]. Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1 ISSN 1064-3745 E-ISBN 978-1-59745-304-2 Library of Congress Control Number: 2007924172
- 7. Dedication To my parents and gurus v
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- 9. vii Preface To meet the emerging needs of genomics, proteomics, and the other omics, microarrays have become unique and important tools for high-throughput analysis of biomolecules. Microarray technology provides a highly sensitive and precise technique for obtaining information from biological samples. It can simultaneously handle a large number of analytes that may be processed rapidly. Scientists are applying microarray technology to understand gene expression, to analyze mutations and single-nucleotide polymorphisms, to sequence genes, and to study antibody–antigen interactions, aptamers, carbohydrates, and cell functions, among many other research subjects. The objective of Microarrays is to enable the researcher to design and fabricate arrays and binding studies with biological analytes. An additional goal is to provide the reader with a broader description of microarray technology tools and their potential applications. In this edition, Microarrays is divided in two parts: Volume 1 deals with methods for preparation of microarrays, and Volume 2 with applications and data analysis. Various methods and applications of microarrays are described and accompanied by exemplary protocols. Volume 2 also covers topics related to bioinformatics, an important aspect of microarray technologies because of the enormous amount of data coming out of microarray experiments. Together, the two volumes provide useful information to the novice and expert alike. Volume 2: Applications and Data Analysis is dedicated to describing applications of microarrays in DNA and protein studies, and for other biomolecules. Several chapters also focus on data analysis and bioinformatics. Chapter 1 covers the applications of microarrays in nonmammalian vertebrate systems and also provides some of the general steps involved in understanding the microarray process. Chapters 2–4 explain the process for selecting the appropriately coated glass slides for coupling of biomolecules and hybridization protocols. Chapter 5 describes the detection of bacterial pathogens using an oligonucleotide microarray format generated from the hydrolysis of PCR probe sequences. Information about genomic copy number changes in a human cancer cell line and analysis by microarray technology is illustrated in Chapter 6. Chapter 7 explains the interactions of biological sample parameters during microarray experiments. Chapter 8 describes the preparation of clinical samples for microarray study, e.g., preparation of nucleic acids from
- 10. frozen and formalin-fixed paraffin-embedded human tissues using macro- and microdissection. Understanding microRNA gene expression in tissues, organs, and cell lines in eukaryotes is discussed in Chapter 9. Genotyping using minisequencing by arrayed primer extension (APEX) and printed arrays extended by a single-nucleotide base is discussed in Chapter 10. A protein chip method for single-base mutation determination in rpoB gene (from Mycobacterium tuberculosis) with high specificity is described in Chapter 11. Chapter 12 relates cDNA library construction by suppression subtraction hybridization. A simple data analysis pipeline using “linear models for microarray data” is discussed, which may be useful for studies of non-model organisms for which there is little genome sequence information. Target preparation methods using a fully integrated ArrayPlex® system based on Biomek FX and cDNA array hybridization using Affymetrix GeneChip® is discussed in Chapter 13. Chapter 14 describes the application of Affymetrix GeneChip® to extraction of mRNA from stimulated and unsitimulated neoplastic and fibroblastic stromal cells for cDNA array hybridization. Application of protein array (ProtoArrayTM) for profiling small molecules is discussed in Chapter 15. Monitoring of clinically relevant markers and regulatory pesticides by microarray is described in Chapter 16. Nanoengineered three-dimensional polyelectrolyte thin films coated glass slides used for the preparation of protein microarrays for detection of cytokine analytes is described in Chapter 17. In Overprinting, Chapter 18, printing is performed by contact and non-contact; it is demonstrated by microarray-based immunoassays without the need for wells or other fluid barriers. It represents about a 1000- fold reduction in consumption of reagents from that for conventional 96-well microtiter plate assay. A microarray based on general principal of microfluidic technology is presented in Chapter 19. In Chapter 20, Ciphergen ProteinChip® Array in combination with Protein Biological System 11C mass spectrometer was applied in analyzing SARS patient samples. Analysis of the data was performed by using Ciphergen ProteinChip® Software. Mass spectrometry procedure is applied in Chapter 21 for high-throughput analysis of affinity protein captured analytes. Linder reviews neural networks, including multiple ANN, in Chapter 22. De Bruyne has explained the typical workflow, error handling, PCA, SOM, and K-nearest neighbor related to microarray data analysis in Chapter 23. The in situ array hybridization thermodynamics, surface density of probes for predicting melting curve parameters is discussed in Chapter 24. Application of Cluster 3.0 and Java Treeview for microarray data clustering, and BGSSJ for functional classification is summarized in Chapter 25. Application of Perl script for designing various probes is describes in Chapter 26. Chapter 27 deals with the integration of array data with sequence, interaction, localization, and other parameters. viii Preface
- 11. I believe this volume, Applications and Data Analysis, will provide valuable information to scientists at all levels, from novice to those intimately familiar with array technology. I would like to thank all the contributing authors for providing manuscripts. My thanks are also due to colleagues for their help in completing this volume. I thank John Walker for editorial guidance and the staff of Humana Press for making it possible to include a large body of available microarray technologies in this volume. Finally, my thanks to my family, especially to my sweet wife Sushma Rampal, for providing all sorts of incentives to complete this project successfully. Jang B. Rampal Preface ix
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- 13. xi Contents Dedication ........................................................................................................v Preface ........................................................................................................... vii Contents of the Companion Volume ............................................................... xv Contributors ..................................................................................................xvii 1 The Use of Microarray Technology in Nonmammalian Vertebrate Systems Conor W. Sipe and Margaret S. Saha ................................................... 1 2 Quality Considerations and Selection of Surface Chemistry for Glass-Based DNA, Peptide, Antibody, Carbohydrate, and Small Molecule Microarrays Jens Sobek, Catharine Aquino, and Ralph Schlapbach ...................... 17 3 Optimization Workflow for the Processing of High Quality Glass-Based Microarrays: Applications in DNA, Peptide, Antibody, and Carbohydrate Microarraying Jens Sobek, Catharine Aquino, and Ralph Schlapbach ...................... 33 4 Processing Protocols for High Quality Glass-Based Microarrays: Applications in DNA, Peptide, Antibody, and Carbohydrate Microarraying Jens Sobek, Catharine Aquino, and Ralph Schlapbach ...................... 53 5 Specific Detection of Bacterial Pathogens Using Oligonucleotide Microarrays Generated From Hydrolysis PCR Probe Sequences Philip J. R. Day .................................................................................... 67 6 Uses of Microarray Platforms in Cancer: A Correlative Study Between Genomic Copy Number Changes and Their Expression at mRNA and Protein Levels Fahd Al-Mulla and Raba Al-Tamimi.................................................... 77 7 Microarray Technology for Use in Molecular Epidemiology Suzanne D. Vernon and Toni Whistler ............................................... 97 8 Utilization of Microarray Platforms in Clinical Practice: An Insight on the Preparation and Amplification of Nucleic Acids From Frozen and Fixed Tissues Fahd Al-Mulla.................................................................................... 115
- 14. xii Contents 9 A Microarray-Based Method to Profile Global microRNA Expression in Human and Mouse Ranjan J. Perera ................................................................................ 137 10 Genotyping of Single-Nucleotide Polymorphisms by Arrayed Primer Extension Scott J. Tebbutt ................................................................................. 149 11 Protein Chip for Detection of DNA Mutations Xian-En Zhang and Li-Jun Bi ............................................................. 163 12 Screening of cDNA Libraries on Glass Slide Microarrays Dave K. Berger, Bridget G. Crampton, Ingo Hein, and Wiesner Vos ........................................................................... 177 13 ArrayPlex SA: A Turn-Key Automated Gene Expression Target Preparation System Handy Yowanto ................................................................................ 205 14 Tumor–Stroma Interactions of Metastatic Prostate Cancer Cells Lines: Analyses Using Microarrays Nicolas Wernert, Annette Kaminski, El-Mustapha Haddouti, and Jens Claus Hahne ................................................................... 223 15 Identification of Small Molecule Targets on Functional Protein Microarrays Michael Salcius, Gregory A. Michaud, Barry Schweitzer, and Paul F. Predki ......................................................................... 239 16 Quantification of Small Molecules Using Microarray Technology Martin Dufva and Claus B. V. Christensen ....................................... 249 17 Antibody-Microarrays on Hybrid Polymeric Thin Film-Coated Slides for Multiple-Protein Immunoassays Xichun Zhou and Jizhong Zhou ........................................................ 259 18 Overprint Immunoassay Using Protein A Microarrays Robert S. Matson, Raymond C. Milton, Jang B. Rampal, Tom S. Chan, and Michael C. Cress ............................................. 273 19 μParaflo™ Biochip for Nucleic Acid and Protein Analysis Qi Zhu, Ailing Hong, Nijing Sheng, Xiaolin Zhang, Anna Matejko, Kyu-Yeon Jun, Onnop Srivannavit, Erdogan Gulari, Xiaolian Gao, and Xiaochuan Zhou ............................................... 287 20 Application of ProteinChip Array Profiling in Serum Biomarker Discovery for Patients Suffering From Severe Acute Respiratory Syndrome Timothy T. C. Yip, William C. S. Cho, Wai-Wai Cheng, Johnny W. M. Chan, Victor W. S. Ma, Tai-Tung Yip, Christine N. B. Lau Yip, Roger K. C. Ngan, and Stephen C. K. Law ................................................................. 313
- 15. Contents xiii 21 Volumetric Mass Spectrometry Protein Arrays Dobrin Nedelkov, Urban A. Kiernan, Eric E. Niederkofler, Kemmons A. Tubbs, and Randall W. Nelson ................................ 333 22 Microarray Data Classified by Artificial Neural Networks Roland Linder, Tereza Richards, and Mathias Wagner .................... 345 23 Methods for Microarray Data Analysis Veronique De Bruyne, Fahd Al-Mulla, and Bruno Pot ..................... 373 24 Predicting DNA Duplex Stability on Oligonucleotide Arrays Arnold Vainrub, Norha Deluge, Xiaolin Zhang, Xiaochuan Zhou, and Xiaolian Gao ............................................. 393 25 Bioinformatics: Microarray Data Clustering and Functional Classification Hsueh-Fen Juan and Hsuan-Cheng Huang ....................................... 405 26 In Silico Gene Selection for Custom Oligonucleotide Microarray Design Conor W. Sipe, Vijay R. Dondeti, and Margaret S. Saha ................. 417 27 Integrated Analysis of Microarray Results Olga G. Troyanskaya ........................................................................ 429 Index ............................................................................................................ 439
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- 17. Contents of the Companion Volume Volume 1: Synthesis Methods 1 Introduction: Array Technology—An Overview Hartmut Seliger 2 Current Microarray Surface Chemistries David W. Grainger, Charles H. Greef, Ping Gong, and Michael J. Lochhead 3 Nonfouling Surfaces: A Review of Principles and Applications for Microarray Capture Assay Designs Ping Gong and David W. Grainger 4 Optimization of Oligonucleotide DNA Microarray Martin Dufva and Claus B. V. Christensen 5 Detection of DNA Copy Number Alterations in Complex Genomes Using Array Comparative Genomic Hybridization Wei-Wen Cai 6 Evaluating the Quality of Data From Microarray Measurements Lili Wang, A. K. Gaigalas, M. B. Satterfield, M. Salit, Y. Zong, and J. Noble 7 Construction of Oligonucleotide Microarrays (Biochip) Using Heterobifunctional Reagents Jyoti Choithani, Bhashyam Vaijayanthi, Pradeep Kumar, and Kailash Chand Gupta 8 Choice of Polymer Matrix, Its Functionalization and Estimation of Functional Group Density for Preparation of Biochips Shweta Mahajan, Bhashyam Vaijayanthi , Gopal Rembhotkar, Kailash Chand Gupta, and Pradeep Kumar 9 Methods in High-Resolution, Array-Based Comparative Genomic Hybridization Mark R. McCormick, Rebecca R. Selzer, and Todd A. Richmond 10 Design and Fabrication of Spotted Long Oligonucleotide Microarrays for Gene Expression Analysis Cheng-Chung Chou and Konan Peck xv
- 18. xvi Contents of the Companion Volume 11 Constructin of In Situ Oligonucleotide Arrays on Plastic Jang B. Rampal, Peter J. Coassin, and Robert S. Matson 12 Detecting Ligated Fragments on Oligonucleotide Microarrays: Optimizing Chip Design, Array Multiplex Ligation-Dependent Probe Amplification Modification, and Hybridization Parameters Ian R. Berry, Carol A. Delaney, and Graham R. Taylor 13 Detection of Single-Nucleotide Polymorphisms in Cancer-Related Genes by Minisequencing on a Microelectronic DNA Chip Alexandre Ho-Pun-Cheung, Hafid Abaibou, Philippe Cleuziat, and Evelyne Lopez-Crapez 14 Hybridization Analysis Using Oligonucleotide Probe Arrays Robert S. Matson and Jang B. Rampal 15 In Situ Synthesis of Peptide Microarrays Using Ink-Jet Microdispensing Bogdan V. Antohe and Patrick W. Cooley 16 Intein-Mediated Peptide Arrays for Epitope Mapping and Kinase/Phosphatase Assays Ming-Qun Xu, Inca Ghosh, Samvel Kochinyan, and Luo Sun 17 Printing Low Density Protein Arrays in Microplates Robert S. Matson Raymond C. Milton, Michael C. Cress, Tom S. Chan, and Jang B. Rampal 18 Forward-Phase and Reverse-Phase Protein Microarray Yaping Zong, Shanshan Zhang, Huang-Tsu Chen, Yunfei Zong, and Yaxian Shi 19 Cell Microarray for Functional Exploration of Genomes David Castel, Marie-Anne Debily, Amandine Pitaval, and Xavier Gidrol 20 Quantification of Mixed-Phase Hybridization on Polymer Microparticles by Europium (III) Ion Fluorescence Kaisa Ketomäki and Harri Lönnberg 21 Measurement of the Sugar-Binding Specificity of Lectins Using Multiplexed Bead-Based Suspension Arrays Kazuo Yamamoto, Fumiko Yasukawa, and Seiichiro Ito 22 Nanotechnology: Moving From Microarrays Toward Nanoarrays Hua Chen and Jun Li
- 19. Contributors FAHD AL-MULLA • Department of Pathology, Molecular Pathology Division, Faculty of Medicine, Kuwait University, Safat, Kuwait RABA AL-TAMIMI • Department of Pathology, Molecular Pathology Division, Faculty of Medicine, Kuwait University, Safat, Kuwait CATHARINE AQUINO • Functional Genomics Center Zurich, Zurich, Switzerland DAVE K. BERGER • Department of Botany, Forestry and Agricultural Biotechnology Institute, University of Pretoria, Pretoria, South Africa LI-JUN BI • Institute of Biophysics, Chinese Academy of Sciences, Beijing, China JOHNNY W. M. CHAN • Department of Medicine, Queen Elizabeth Hospital, Kowloon, Hong Kong TOM S. CHAN • Beckman Coulter, Inc., Fullerton, CA WAI-WAI CHENG • Department of Clinical Oncology, Queen Elizabeth Hospital, Kowloon, Hong Kong WILLIAM C. S. CHO • Department of Clinical Oncology, Queen Elizabeth Hospital, Kowloon, Hong Kong CLAUS B. V. CHRISTENSEN • Department of Micro and Nanotechnology, Technical University of Denmark, Lyngby, Denmark BRIDGET G. CRAMPTON • CSIR Biosciences and African Centre for Gene Technologies, Pretoria, South Africa MICHAEL C. CRESS • Beckman Coulter, Inc., Fullerton, CA PHILIP J. R. DAY • Manchester Interdisciplinary Centre, University of Manchester, Manchester, UK; Analytical Sciences, ISAS, Dortmund, Germany VERONIQUE DE BRUYNE • Applied-Maths BVBA, Sint-Martens-Latem NORHA DELUGE • Department of Biology and Biochemistry, University of Houston, Houston, TX VIJAY R. DONDETI • Department of Cellular and Molecular Biology, University of Pennsylvania, Philadelphia, PA MARTIN DUFVA • Department of Micro and Nanotechnology, Technical University of Denmark, Lyngby, Denmark xvii
- 20. xviii Contributors XIAOLIAN GAO • Department of Biology and Biochemistry, University of Houston, Houston, TX ERDOGAN GULARI • Department of Chemical Engineering, University of Michigan, Ann Arbor, MI EL-MUSTAPHA HADDOUTI • Institute of Pathology, University of Bonn, Bonn, Germany JENS CLAUS HAHNE • Institute of Pathology, University of Bonn, Bonn, Germany INGO HEIN • Scottish Crop Research Institute, Dundee, Scotland, UK AILING HONG • Atactic Technologies Inc., Houston, TX HSUAN-CHENG HUANG • Institute of Bioinformatics, National Yang-Ming University, Taipei, Taiwan HSUEH-FEN JUAN • Department of Life Science, Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan KYU-YEON JUN • Department of Biology and Biochemistry, University of Houston, Houston, TX ANNETTE KAMINSKI • Institute of Pathology, University of Bonn, Bonn, Germany URBAN A. KIERNAN • Intrinsic Bioprobes Inc., Tempe, AZ CHRISTINE N. B. LAU YIP • Ciphergen Biosystems Incorporation, Fremont, CA STEPHEN C. K. LAW • Department of Clinical Oncology, Queen Elizabeth Hospital, Kowloon, Hong Kong ROLAND LINDER • Institute of Medical Informatics, University of Lübeck, Lübeck, Germany VICTOR W. S. MA • Department of Clinical Oncology, Queen Elizabeth Hospital, Kowloon, Hong Kong ANNA MATEJKO • Atactic Technologies Inc., Houston, TX ROBERT S. MATSON • Beckman Coulter, Inc., Fullerton, CA GREGORY A. MICHAUD • Invitrogen Corporation, Branford, CT RAYMOND C. MILTON • Beckman Coulter, Inc., Fullerton, CA FAHD AL-MULLA • Department of Pathology, Molecular Pathology Division, Faculty of Medicine, Kuwait University, Safat, Kuwait DOBRIN NEDELKOV • Intrinsic Bioprobes Inc., Tempe, AZ RANDALL W. NELSON • Intrinsic Bioprobes Inc., Tempe, AZ ROGER K. C. NGAN • Department of Clinical Oncology, Queen Elizabeth Hospital, Kowloon, Hong Kong
- 21. Contributors xix ERIC E. NIEDERKOFLER • Intrinsic Bioprobes Inc., Tempe, AZ RANJAN J. PERERA • Keck Graduate Institute, Claremont, CA BRUNO POT • Applied-Maths BVBA, and Bacteriology of Ecosystems, Institut Pasteur de Lille (IBL), Lille Cedex, France PAUL F. PREDKI • Invitrogen Corporation, Branford, CT JANG B. RAMPAL • Beckman Coulter, Inc., Brea, CA TEREZA RICHARDS • The Library, University of the West Indies, Mona, Kingston, Jamaica, West Indies MARGARET S. SAHA • Department of Biology, College of William and Mary, Williamsburg, VA MICHAEL SALCIUS • Invitrogen Corporation, Branford, CT RALPH SCHLAPBACH • Functional Genomics Center Zurich, Zurich, Switzerland BARRY SCHWEITZER • Invitrogen Corporation, Branford, CT NIJING SHENG • Atactic Technologies Inc., Houston, TX CONOR W. SIPE • Department of Biology, College of William and Mary, Williamsburg, VA JENS SOBEK • Functional Genomics Center Zurich, Zurich, Switzerland ONNOP SRIVANNAVIT • Atactic Technologies Inc., Houston, TX, and Department of Chemical Engineering, University of Michigan, Ann Arbor, MI RABA AL-TAMIMI • Department of Pathology, Molecular Pathology Division, Faculty of Medicine, Kuwait University, Safat, Kuwait SCOTT J. TEBBUTT • James Hogg iCAPTURE Center for Cardiovascular and Pulmonary Research, University of British Columbia, Vancouver, BC,Canada OLGA G. TROYANSKAYA • Lewis-Sigler Institute for Integrative Genomics, Carl Icahn Laboratory, Princeton University, Princeton, NJ KEMMONS A. TUBBS • Intrinsic Bioprobes Inc., Tempe, AZ ARNOLD VAINRUB • College of Veterinary Medicine, Auburn University, Auburn, AL SUZANNE D. VERNON • Division of Viral and Rickettsial Diseases, National Centers for Infectious Diseases, Center for Disease Control and Prevention, Atlanta, GA WIESNER VOS • Department of Statistics, Oxford University, Oxford, UK MATHIAS WAGNER • Department of Pathology, Saarland University, Homburg-Saar, Germany
- 22. NICOLAS WERNERT • Institute of Pathology, University of Bonn, Bonn, Germany TONI WHISTLER • Division of Viral and Rickettsial Diseases, National Centers for Infectious Diseases, Center for Disease Control and Prevention, Atlanta, GA TAI-TUNG YIP • Ciphergen Biosystems Incorporation, Fremont, CA TIMOTHY T. C. YIP • Department of Clinical Oncology, Queen Elizabeth Hospital, Kowloon, Hong Kong HANDY YOWANTO • Beckman Coulter, Inc., Fullerton, CA XIAN-EN ZHANG • Institute of Biophysics, Chinese Academy of Sciences, Beijing, China XIAOLIN ZHANG • Atactic Technologies Inc., Houston, TX JIZHONG ZHOU • Environmental Science Division, Oak Ridge National Lab, Oak Ridge, TN XIAOCHUAN ZHOU • Atactic Technologies Inc., Houston, TX XICHUN ZHOU • Environmental Science Division, Oak Ridge National Lab, Oak Ridge, TN QI ZHU • Department of Biology and Biochemistry, University of Houston, Houston, TX xx Contributors
- 23. 1 From: Methods in Molecular Biology, vol. 382: Microarrays: Second Edition: Volume 2 Edited by: J. B. Rampal © Humana Press Inc., Totowa, NJ 1 The Use of Microarray Technology in Nonmammalian Vertebrate Systems Conor W. Sipe and Margaret S. Saha Summary Among vertebrates, the mammalian systems that are frequently used to investigate questions related to human health have gained the most benefit from microarray technology to date. However, it is clear that biological investigations and the generalized conclusions drawn from them, can only be enhanced by including organisms in which specific processes can be readily studied because of their genetic, physiological, or developmental disposition. As a result, the field of functional genomics has recently begun to embrace a number of other vertebrate species. This review summarizes the current state of microarray technology in a subset of these vertebrate organisms, including Xenopus, Rana, zebrafish, killifish (Fundulus sp.), medaka (Oryzias latipes), Atlantic salmon, and rainbow trout. A summary of various applications of microarray technology and a brief introduction to the steps involved in carrying out a microarray experiment are also presented. Key Words: Atlantic salmon; Fundulus; medaka; microarray; rainbow trout; Xenopus. 1. Introduction The use of microarray technology has resulted in substantial progress in many areas of biology where standard experimental organisms (e.g., mouse, yeast, Caenorhabditis elegans, Drosophila, Arabidopsis) are employed. Among vertebrate species, the mammalian models (predominantly mouse and rat) used to study processes implicated in human health have reaped the most benefit from microarrays to date. A number of other vertebrate models traditionally uti- lized in developmental and toxicological investigations have been somewhat slower to take advantage of array technology, in large part because of having significantly fewer genomic resources available.
- 24. 2 Sipe and Saha Biological investigations and specifically the validity of generalized conclu- sions, can only be enhanced by using a diversity of species in which specific phys- iological, developmental, or biochemical traits are readily studied (1). Moreover, a better understanding of the genes effecting adaptive changes and leading to pheno- typic differences can be gained by exploring gene expression profiles in other ver- tebrates for which a wealth of ecological, phenotypic, and genetic data are available (2). Along these lines, functional genomics has recently branched out to a number of other vertebrate systems that might be more suitable for addressing particular questions in a variety of fields. This review will endeavor to: (1) sum- marize the current state of microarray technology in a subset of these vertebrate organisms that includes Xenopus, Rana, zebrafish, killifish (Fundulus sp.), medaka (Oryzias latipes), Atlantic salmon, and rainbow trout and (2) provide a brief intro- duction to the steps involved in carrying out a microarray experiment for investi- gators interested in introducing this technique into their laboratories. 2. Overview of Applications 2.1. Development A primary goal of developmental biology is to understand the underlying net- works of gene and protein interactions that control the processes leading to the final body plan of an organism (3). However, this goal is technically challenging when using traditional molecular techniques, which rely on examining a limited number of genes in a given experiment (e.g., in situ hybridization), although extensive and informative analyses have been performed using these methods (4–6). The advent of microarray technology has allowed the profiling of thou- sands of transcripts simultaneously and has given investigators the ability to examine large-scale changes in gene expression over the course of the develop- ment. The utility of this technique is demonstrated by the marked increase in the number of microarray investigations in two nonmammalian vertebrates, Xenopus and zebrafish, over the past year (a total of 19 at the time of writing). These microarray experiments can be roughly divided into two categories, the first of which seeks to determine gene expression profiles at different points in normal development. The expression information obtained from such general surveys can suggest coexpressed gene clusters, identify region- or tissue-specific molecular markers, and make predictions regarding gene function. Recently, the first large-scale analysis of gene expression in the Xenopus embryo was reported (7). Likewise, studies in other systems have established expression profiles for embryos at different developmental stages in zebrafish (8–10) and medaka (11). More specific developmental processes have also been examined using microar- rays, such as the maturation and development of trout ovary (12), the differences between maternal and zygotic contribution to the embryonic transcriptome in Xenopus (13), and the identification of Xenopus oocyte-specific transcripts (14).
- 25. Another class of studies examines the changes in gene expression brought about by experimental perturbation of a specific developmental pathway. Following perturbation of normal gene expression, genes that are coordinately up- or downregulated can be identified and used to construct molecular path- ways that aid in the prediction of the roles of genes with unknown function. In Xenopus and zebrafish, the two best-characterized organisms in the vertebrates considered in this review, this has been achieved using a number of molecular biology techniques. Most commonly, a specific protein or pathway is inhibited with chemical inhibitors or agonists, as in the case of the Xenopus FGF (15), retinoic acid (16), and BMP2 pathways (17). Likewise, microinjection into embryos can be used to manipulate a given pathway through a dominant nega- tive receptor mRNA (18), injection of a misexpressed transcript (19,20), or through morpholino-based knockdown of gene function (21). The less techni- cally demanding, yet equally effective, approach of physical amputation has been successfully applied in a large-scale analysis of fin regeneration in the adult medaka (22). The previously described studies demonstrate the power of the microarray when combined with the molecular perturbations and/or other clas- sical embryological techniques (i.e., explants, rotation operations, lineage trac- ing, and so on) available in these nonmammalian vertebrates. 2.2. Physiology A number of studies in fish systems have investigated the transcriptional response to various physiological challenges. Microarray technology has proven a useful tool for dissecting the molecular foundations of host–microbial interactions. Molecular biomarkers for infection in the Atlantic salmon have been proposed based on the profile of genes upregulated in response to two common pathogens of the Atlantic salmon (23–25). Similarly, zebrafish reared in a sterile environment have implicated genes regulated by microbiota of the gut, including those involved in epithelial proliferation, promotion of nutrient metabolism, and innate immune responses (26). The fish also provides an excellent system to answer more traditional phys- iological questions using microarrays. Combining a hypothesis-driven and discovery-based approach, examination of the transcriptional response was reported as a result of the large variations in temperature the killifish experiences on both daily and seasonal bases (27). Unsurprisingly, the control of cell growth and proliferation are an important part of the response to temperature change; however, they report these pathways are regulated by different batteries of genes in constant against the fluctuating temperatures. Chronic temperature reduction in skeletal muscle from adult zebrafish results in a similar pattern of gene reg- ulation when compared with Fundulus (28). In addition, the transcriptional responses to hypoxia and physical stress, two physiological states having clear Microarrays in Nonmammalian Vertebrates 3
- 26. implications for human health and disease have been characterized in zebrafish (8,29) and trout (30). 2.3. Toxicology To date, classical model organisms have gained the most benefit from microarray research, owing mostly to the immense knowledge already accumu- lated in these systems. However, these organisms are rarely the most relevant for use in environmental biology and toxicology (31). Fish species, on the other hand, are important biomonitoring tools for toxicologists, as aquatic ecosys- tems are the major recipient of human-produced pollutants (32). As a result, many groups have begun using newly developed microarrays to measure the effects of contaminants on these species. Koskinen (33) endeavored to construct a rainbow trout cDNA microarray for use in toxicological studies and tested its efficacy by exposing trout fry to four model aquatic contaminants. Likewise, the effects of exposure to sublethal concentrations of zinc were studied in trout using a cross-species Fugu ruprides gill cDNA filter array (31). In addition to those present in fish models, a standardized test to determine the relative developmental toxicity hazard of chemical agents is already in place in Xenopus (frog embryo teratogenesis assay). This system has been used extensively to minimize the cost and time constraints associated with the use of conventional mammalian test organisms (34). The power of the frog embryo teratogenesis assay toxicological testing system could be increased dramati- cally by using recently designed Xenopus microarrays in conjunction with purely morphological assays. 2.4. Evolution, Comparative Genomics, and Ecology Spurred on by the success of functional genomics in the areas of develop- ment and physiology, investigators have sought to apply microarrays in the investigation other diverse biological processes (35,36). Oleksiak et al. (1) have attempted to estimate the degree of natural variation in gene expression within and between natural populations of Fundulus. An initial study showed a rela- tively large number of genes in members of the same population often varied by a factor of 1.5 or more (37). A more recent investigation found an associa- tion between cardiac metabolism and mRNA expression profiles, again sug- gesting that subtle variations in gene expression exist between individuals (38). Other groups have attempted to examine the conservation of molecular path- ways between species by using heterologous mRNA in microarray hybridiza- tions. This approach has proved feasible in Xenopus (39), zebrafish and cichlid fishes (2), and salmonids (31,40). Microarray technology has also been used to elucidate the molecular underpinnings of observed ecological phenomena For instance, Mori (41) recently examined the molecular basis of predator- induced morphological changes used as a defensive strategy in Rana tadpoles. 4 Sipe and Saha
- 27. 3. Microarray Design The two most widely used microarray systems are long oligonucleotide and cDNA microarrays. Long oligonucleotide microarrays are generated by chem- ically synthesizing oligonucleotides (usually 40–70 bp in length) and affixing them to slides, while cDNA microarrays are created by spotting long strands of amplified cDNA sequences. 3.1. cDNA Arrays Many of the earlier-mentioned references have successfully utilized cDNA microarrays in experiments. This strategy typically relies on randomly gener- ated libraries of expressed sequence tags (ESTs) to dictate the number and makeup of probe sequences included on a given chip. While subtractive methods have been applied to enrich EST libraries to select for transcripts from specific structures (12,14,40), the final sequence representation on most chips remains relatively indiscriminate. A different approach to selecting ESTs for inclusion on a microarray is to base it on membership in gene-oriented sequence clusters, such as the National Center for Biotechnology Information (NCBI) unigene database (42,43). However, this method is hampered by an unsatisfactory level of sequence annotation, as numerous ESTs from these projects have not been assigned an identity based on homology (7,18). Although, some of these unan- notated sequences might represent species-specific genes, they are more likely unidentifiable owing to a lack of extensive genomic resources in these organ- isms. These issues will certainly be resolved in the future as more sequencing resources are applied to these nonmammalian vertebrates. One advantage that cDNA arrays offer over oligonucleotide-based chips is the ready access they provide to cDNAs with which to perform whole-mount in situ hybridization analysis for independent evaluation of data. Microarrays are available for some of the species under consideration in this review from various research groups: two different salmon microarrays (4000 and 16,000 spots; also used for rainbow trout hybridizations) are available from the Genomic Research onAtlantic Salmon project (http://web.uvic.ca/cbr/grasp/) and a 5000 feature Xenopus cDNA from the Xenopus Microarray Project (http:// silico.ucsd.edu/xenopus/). Other cDNA arrays can be acquired by contacting the corresponding authors in individual papers, who are usually willing to dis- tribute arrays for a nominal fee to cover expenses. 3.2. Long Oligonucleotide As a result of the effort associated with constructing cDNA arrays, the use of long oligonucleotide arrays has become increasingly popular as an alternative platform. It has even been suggested that oligonucleotide microarrays are more reliable for determining changes in gene expression than their cDNA counter- parts (44). A major drawback of these arrays is their associated costs, as the Microarrays in Nonmammalian Vertebrates 5
- 28. chemically synthesized oligonucleotide probes are usually acquired from a commercial source. Currently, there are pre-made oligonucleotide sets and microarrays available for Xenopus (Operon, www.operon.com), zebrafish (Operon; Agilent, www.agilent.com; Sigma-Genosys, www.sigma-genosys.com; Ocimum Biosolutions, www.ocimumbio.com), and medaka (Kimura et al. report their array will be available from Agilent in the future). With the increased prevalence of custom microarray fabrication services, it has also become possible for investigators to design microarrays tailored to answer specific questions or investigate gene expression patterns in organisms for which commercial arrays do not exist. However, the high probe densities of oligonucleotide arrays make a manual selection of the genes included on a given chip, which is otherwise an impractical task. To overcome this problem, in silico probe selection method has recently been described that is applicable for any organism having sequence data (45). This strategy relies on using pub- licly available microarray information to interrogate existing sequence data and identify a set of homologous genes in the organism of interest, and is particu- larly suitable for designing microarrays to investigate processes that are con- served among species. The large amounts of data made available by the large EST sequencing consortiums and also the rapid progress of various sequencing initiatives, make these nonmammalian vertebrates ideal candidates for the application of this microarray design method. 4. Experimental Design and Methods The following is a general survey of methods involved in the major steps in a microarray experiment with no single topic treated in exhaustive detail. It is intended to be an introduction to those investigators who wish to begin the use of microarrays in their laboratories. 4.1. General Considerations Ultimately, the specific question being asked is the most important consider- ation when designing an effective microarray experiment. This will generally dictate the type and amount of material available with which to work, as well as the most important comparisons to address the question being posed. A major decision is whether to use a direct (i.e., experimental vs control) or indirect (i.e., experimental vs reference, control vs reference) comparison of expression values. Pairwise comparisons between all samples will yield the most precise estimates of transcriptional differences (46), and for many types of experiments, such as evaluating the effect of a toxin (33), the perturbation of a specific pathway (16,18), or comparing the disease state with the normal (23,25), a direct comparison is the logical choice. When performing direct com- parisons, most investigators have opted for dye-swap replications, where the 6 Sipe and Saha
- 29. hybridization conditions are repeated with dye assignments reversed in the sec- ond hybridization in order to minimize systematic bias introduced by differ- ences in the intensities of the two dyes. A major drawback of this design is that it becomes increasingly less feasible in terms of cost and materials as more samples are added to an experiment. Consequently, for experiments that measure differences in a large number of samples (e.g., over a developmental time-course) (7,29) or repeated meas- urements of fluctuating phenomena (27), a reference design, where each experimental sample is compared with a common reference, is often used. The indirect expression values obtained from such a reference design have been shown to be robust when directly compared with those from all-pair experi- mental design (47). Ideally, a reference sample should represent the genes expressed in all samples and with intermediate expression levels (7). The abundance of amphibian and fish embryos that can be obtained and their dis- crete staging lends itself to the creation of such a pool. Additionally, the use of a universal reference sample for a given organism facilitates the compari- son of microarray data sets generated in different experiments, which is oth- erwise an impractical task. Initial steps to define a common reference pool of RNA have been taken in Xenopus, where RNA from eight embryonic samples has been used (7,47). In order to correct for subtle variation between differ- ent preparations of a reference RNA pool, it is necessary to fluorescently label each batch and compare the resulting signal intensities in a pairwise hybridization. 4.2. RNA Isolation and Amplification Pure RNA preparations are critical to ensure reliable gene expression results. Partially degraded RNA can bias cDNA labeling toward sequences that lie near the 3′ terminus and therefore, might distort the relative proportions of various RNA species when hybridized. To preclude this, many investigators use the Agilent BioAnalyzer (Agilent Technologies, Palo Alto, CA), an instrument capa- ble of visualizing picogram amounts of material, to measure RNA quality. Total RNA isolation techniques can roughly be divided into two categories represented in approximately equal proportions in the literature: (1) those that use phase sepa- ration to sequester RNA from proteins and DNA (e.g., Invitrogen’s TRIzol [Invitrogen, Carlsbad, CA]) or (2) column-based chromatography strategies (e.g., Qiagen RNeasy, [Qiagen, Valencia, CA] Ambion MegaClear [Ambion, Austin, TX]). The use of both techniques, which fractionate RNA on a different physical basis, in conjunction provides significantly better purification than either method performed independently (46). In some experimental situations, it is impossible to isolate the substantial amounts of total RNA (40–60 μg) needed for a typical labeling reaction. Such Microarrays in Nonmammalian Vertebrates 7
- 30. is the case when working with tissue from embryonic explants, portions of organs, or other small populations of cells. To obtain the necessary template for target labeling, several RNA amplification methods have been developed. The predominant procedure relies on using a bacteriophage RNA polymerase to transcribe a cDNA template into aRNA (antisense RNA). This transcription is accomplished by synthesizing single-stranded cDNA from experimental RNA using primers containing a synthetic SP6, T3, or T7 RNA polymerase promoter. The final aRNA target has been demonstrated to reflect accurately the size, complexity, and abundance of mRNAs in the original RNA population (48,49). Several commercial kits are available that implement this method (e.g., MessageAmp II, Ambion; RiboAmp, Arcturus [Sunnyvale, CA]; Superscript system, Invitrogen; BioArray system, Enzo Life Sciences [Farmingdale, NY]). 4.3. cDNA Labeling Investigators are again presented with multiple options when preparing a labeled target solution for hybridization to a microarray. To date, most investiga- tors have chosen to use the cyanine-based dyes Cy3 and Cy5 (GE Healthcare, Piscataway, NJ) to label cDNA; however, a range of alternative dyes (e.g., Alexa Fluor dyes, Invitrogen) are available that are reported to be stronger, more resist- ant to photobleaching, and less sensitive to pH changes (50). Regardless of flu- orophore choice, labels can be incorporated into cDNA directly through the inclusion of dye-conjugated nucleotides or through a coupling reaction between a reactive dye and amine-substituted nucleotides. Direct labeling techniques are hampered by the ability of reverse transcriptase to incorporate bulky dye-conjugated nucleotides, resulting in labeled cDNA of low fluorescent intensity and a subsequent reduction in assay sensitivity. Nevertheless, many commercial enzymes have been engineered to improve incorporation of fluorophores (e.g., SuperScript III, Invitrogen; CyScribe, GE Healthcare; Omniscript, Qiagen), and many investigators have chosen to utilize this method as it is cost efficient and requires fewer steps. Indirect labeling relies on amino-allyl nucleotides (which are incorporated as efficiently as unmodified nucleotides) that are conjugated to an ester-linked dye molecule following cDNA synthesis. Indirect labeling generally results in a higher overall level of cDNA fluorescence by greatly reducing enzymatic bias, though is a more involved process. The efficiency and yield of a dye labeling reaction is measured using stan- dard spectroscopy, where the absorbance of the nucleic acid at 260 nm and the absorbance of the dye at its emission maximum are measured. Because the vol- umes involved are generally quite small, these measurements are usually taken in microcuvettes (10–200 μL) or spectrophotometers specifically designed for small sample volumes (e.g., the Nanodrop, Nanodrop Technologies). 8 Sipe and Saha
- 31. 4.4. Hybridization Conditions Individual hybridization conditions will vary depending on the type of microarray employed in a given assay. The majority of commercial microarrays are distributed with detailed protocols containing a suggested hybridization regime. In general, custom arrays are hybridized under the same conditions of high stringency to deter nonspecific cross-hybridizations (46). Besides labeled target cDNA, typical hybridization solutions contain buffers that are of high ionic strength to reduce electrostatic repulsion and promote complementary base pairing, as well as blocking agents and/or detergents to minimize back- ground. The majority of hybridizations in the references reviewed earlier have also included deionized formamide (30–50%) in hybridization solu- tions. Formamide lowers the melting temperature of hybrids, thus allowing the temperature of hybridization to be lowered. This reduces the opportunity for target solution evaporation, which can occur at the higher temperatures needed for aqueous hybridizations. 4.5. Washing Posthybridization washing is another critical step that has the potential to affect the quality of expression data obtained. Inadequate wash conditions will lead to splotches of high background fluorescence in the final scanned image, making detection of genuine signal impossible. Although many com- mercial arrays come with standard washing protocols, the optimal wash condi- tions for a particular set of targets should be determined empirically. Generally, these will consist of successively weaker SSC washes (e.g., from 2X to 0.1X) to remove all traces of the target solution, which contains unhybridized cDNA molecules and other substances (e.g., SDS) that can contribute to background fluorescence (46). A common error to be avoided is allowing the slide to dry (even partially) during the washing procedure, as this will lead to a deposi- tion of salt and SDS on the surface of the array that is difficult to remove. Liberal agitation and repeated dunking of slides during these washes is often required to avoid high amounts of irregular background fluorescence. If, after scanning an array, high background fluorescence interferes with signal, the washing steps can be repeated at a higher stringency (i.e., higher temper- ature, longer incubation times, or more agitation) to remove all traces of hybridization solution. 4.6. Image Acquisition The importance of the image acquisition step cannot be overstated, as all subsequent data analyses follow from it. Scanning presents the challenge of optimizing a number of parameters to obtain the best possible image in the Microarrays in Nonmammalian Vertebrates 9
- 32. fewest number of scans. Some of these parameters vary depending on both array type and scanner manufacturer and they need to be set before the final image acquisition begins; these can include focal length, scan resolution, and scan speed. Common to all arrays, however, is the need to maximize spot inten- sity over that of background. In an ideal microarray image, the intensity of spots corresponding to the lowest-expressed genes should lie just above background pixel intensities, and the intensities of the highest-expressed genes just below saturation levels (i.e., the point at which pixel intensity falls outside the instru- ment’s dynamic range of detection). The majority of microarray scanners presently available (e.g., Perkin- Elmer ScanArray, Axon GenePix scanners) rely on confocal optics with one or more lasers as excitation sources to scan spots in a pixel-by-pixel manner. In these systems, photomultiplier tubes (PMT) detect photons emitted by labeled cDNA hybridized on the microarray and change the signal into an electrical current that is converted, in turn, to discrete digital values. Thus, two main scanning parameters increase or decrease the spot signal intensities obtained in a given scan: the power of the excitation source (i.e., laser power) and the gain of the PMT. As a rule, PMT gain should be increased before raising laser power, as higher laser energies will often lead to rapid photo- bleaching of the array. On the other hand, PMT gain indiscriminately raises the intensity of all pixels and magnifies background noise as well as true sig- nal. Frequently, a coordinate adjustment of both parameters is required to ensure that as many spots as possible lie in between the two extremes of sat- uration and background. 4.7. Data Analysis Spot intensities in microarray image files are quantified using any one of a multitude of commercially available software packages (e.g., GenePix Pro, Axon; ScanArray Express, Perkin-Elmer; Imagene, Biodiscovery). A com- puter file (most commonly a .gal file, provided with commercial arrays or cre- ated by spotting software) provides a link between the scanned image and the identities of the genes or sequences at a given position on the microarray. Generally, in the first step of data analysis, background-corrected median or mean spot intensity values are calculated, and features of poor quality are filtered out. Individual software packages use different parameters to flag poor quality spots, but all rely on some combination of morphology, number of saturated pixels, or overall intensity. Using a Xenopus array, a simple and robust method is developed for determining true spot signal using mean to median correlation (51). Those spots that survive this initial filtering step are normalized to remove intensity- and position-dependent bias in the quantifica- tion of each feature (most commonly using the locally weighted scatterplot 10 Sipe and Saha
- 33. smoothing [LOESS] method), and a final ratio of intensities (experimental/reference or experimental/control) is calculated for each spot. 4.7.1. Pattern Discovery Numerous techniques have been developed to aid researchers in discovering and visualizing patterns present in gene expression data generated by microar- ray experiments (52). In general, these techniques are excellent choices for ini- tial data analysis, as they can suggest gene interactions or members of functional pathways (46). Probably the most prevalent of these computational methods is cluster analysis, which constructs relationships based on expres- sion patterns observed in data sets as a whole (53,54). Both treatment groups and genes can be clustered by some measure of similarity (which vary by method) to organize samples together into familiar tree-like dendograms. Those genes (or groups having genes), which are up- or downregulated in a roughly similar manner lie closest to one another in the tree. Another family of pattern discovery techniques, which includes principal component analysis and multidimensional scaling, strives to reduce the number of variables needed to represent a data set while retaining a maximum amount of its variability (55). The most informative (i.e., those contributing the most variability to the dataset as a whole) variables are then projected into two- or three-dimensional space, and can separate treatments or gene clusters into revealing groups. 4.7.2. Statistical Analysis The statistical treatment of microarray data has been extensively treated in the literature (for detailed reviews, see refs. 52,56 and 57). Until relatively recently, fold changes in gene expression were widely used to select candidate genes from a data set for further study (as in ref. 13). However, this approach has been the object of criticism, as it does not give a measure of how signifi- cant such a difference is likely to be (57). As a result, a number of different sta- tistical tests have been utilized in the previously listed references to detect significantly differentially expressed genes in microarray data sets, including the Students’ t-test (21,29,30,33), F-test (10), analysis of variance (37,38,58), and Bayesian analyses (16). Another statistical method, Significance analysis of microarrays, was developed by Tusher (59) specifically for microarrays and has gained widespread acceptance in the literature (14,25,39). 4.7.3. Independent Confirmation of Results It is important to note that the data obtained from a single microarray exper- iment will only implicate genes as candidates for involvement in a given net- work or pathway. It is up to the investigator to decide whether the results are accurate for the particular biological system under study (60). Consequently, Microarrays in Nonmammalian Vertebrates 11
- 34. other experimental methods are used to validate independently the gene expres- sion levels measured using a microarray. The exact technique used will vary based on the scientific question, but commonly used techniques include semi- quantitative reverse transcription polymerase chain reaction (RT–PCR), quanti- tative real-time PCR, Northern blot, ribonuclease protection assay, in situ hybridization, or immunohistochemistry. Real-time PCR is the choice of many for acquiring precise measurements of candidate gene expression levels, as the method is rapid and relatively simple to perform once established in a laboratory. 5. Online Information Sources 5.1. Genome Sequencing 1. Danio rerio Sequencing Project at the Sanger Institute, http://www.sanger.ac.uk/ Projects/D_rerio/. 2. JGI Xenopus tropicalis genome project, http://genome.jgi-psf.org/Xentr4/Xentr4. home.html. 3. Salmon Genome Project, http://www.salmongenome.no/cgi-bin/sgp.cgi. 4. Medaka Genome Project, http://dolphin.lab.nig.ac.jp/medaka/. 5. NBRP Medakafish Genome Project, http://shigen.lab.nig.ac.jp/medaka/genome/ top.jsp. 5.2. EST Sequencing 1. Xenopus, zebrafish, salmon, trout, and killifish Gene Indices at TIGR, http://www.tigr.org/tdb/tgi/index.shtml. 2. Washington University Zebrafish EST sequencing project, http://www.genetics. wustl.edu/fish_lab/frank/cgi-bin/fish/. 3. Xenopus tropicalis EST project at the Sanger Institute, http://www.sanger.ac.uk/ Projects/X_tropicalis/. 4. BLAST interface to search the GRASP EST database, http://snoopy.ceh.uvic.ca/. 5. FunnyBase, a database of functional information for Fundulus ESTs, http://genomics. rsmas.miami.edu/funnybase/super_craw4/. 6. MeBase, a database of medaka ESTs, http://mbase.bioweb.ne.jp/~dclust/ me_base.html. 5.3. Other Sites of Interest 1. The Zebrafish Information Network, http://www.zfin.org. 2. A web-based Xenopus resource, http://www.xenbase.org. 3. Genomic Research on Atlantic Salmon Project, http://web.uvic.ca/cbr/grasp/. 4. The main medaka web resource site, http://biol1.bio.nagoya-u.ac.jp:8000/. 5. The official NCBI Handbook, http://www.ncbi.nlm.nih.gov/books/bv.fcgi?call=bv. View.ShowTOC&rid=handbook.TOC&depth=2. 6. Y.F. Leung’s huge database of microarray links, http://ihome.cuhk.edu.hk/~b400559/ array.html. 12 Sipe and Saha
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- 39. 17 From: Methods in Molecular Biology, vol. 382: Microarrays: Second Edition: Volume 2 Edited by: J. B. Rampal © Humana Press Inc., Totowa, NJ 2 Quality Considerations and Selection of Surface Chemistry for Glass-Based DNA, Peptide, Antibody, Carbohydrate, and Small Molecule Microarrays Jens Sobek, Catharine Aquino, and Ralph Schlapbach Summary The complexity of workflows for the production of high quality microarrays asks for the care- ful evaluation and implementation of materials and methods. As a cornerstone of the whole microarray process, the microarray substrate has to be chosen appropriately and a number of cru- cial considerations in respect to matching the research question with the technical requirements and possibilities have to be taken into account. In the following, how to lay the fundamental for high performance microarray experiments by evaluating basic quality requirements and the selec- tion of suitable slide surface architectures for a variety of applications was concentrated. Key Words: Microarrays; quality; surface chemistry; spot morphology; immobilization. 1. Introduction Microarrays have become an indispensable and highly efficient tool for the investigation of genome alterations and large-scale gene expression patterns in basic and applied research in the academic and industrial world. Emerging uses of microarrays for the elucidation of protein-binding activity, antibody–epitope specificity, and functional protein enzyme assays, can be foreseen for the near future. The fact that microarray technology is not limited to DNA and protein applications only is well-illustrated by latest developments in the generation and use of carbohydrate arrays for binding studies or cellular on-the-chip assays. In order to cope with the various technical issues connected to the fabrication and use of microarrays in these diverse areas, expertize in many fields of physical and organic chemistry, biochemistry, and molecular biology has to be combined with latest technologies in engineering and bioinformatics. In this article, guide- lines on the selection of a suitable slide surface that is usually dictated by the
- 40. 18 Sobek et al. planned application are provided. In turn, the chosen surface chemistry deter- mines the experimental conditions of immobilization, blocking, and hybridiza- tion, which have to be carefully optimized. Stating this, the principle production parameters have been optimized in a large number of experiments for a variety of slides with different slide chemistries and for diverse applications, such as long and short DNA, peptide, protein, antibody, carbohydrate, and small mole- cule microarrays. As a result, general recommendations have been established on how experimental conditions for custom made microarrays can be opti- mized. This workflow and optimized protocols will be presented in the subse- quent Chapters 3 and 4. 2. Methods 2.1. General Quality Considerations Glass quality and the quality of the coatings determine the optical properties of the slide such as probe immobilization efficiency and spot morphology. Slide quality is a crucial factor in the production of microarrays and is determined by a combination of chemical composition, flatness, autofluorescence, and glass homogeneity. Scratches, deposits, or other artifacts might arise from the pro- duction process of the glass and the subsequent chemical coating. Additionally, abrasion from the plastic packings might leave particles on the slide surface (see Note 1). In order to evaluate the quality of a slide, a quick glance across the surface of the slide held into the light under a small angle reveals any visible chemical depositions, detectable as gray shadows or small islands of speckles. Irrespective of the type of damage, such slides should not be used for microar- ray applications. A second simple but highly efficient quality test is scanning the slide at highest laser intensity and PMT amplification (Fig. 1). A very low- signal intensity of homogeneous distribution should be the basis for rejection of the slide. It is important to note that most of the slides on the market are based on a wet chemical silanation reaction, which is very difficult to perform under controlled conditions. Manufacturers should be selected carefully for such type of slide in order to obtain the best possible results in a microarray experiment regarding reliability, reproducibility, and detection limit. All slide errors must be kept as small as possible. Low quality slides should be rejected as they bear the risk of failing an experiment (see Note 2). The next important parameter in the production of microarrays is the spot- ting process, which often leads to imperfect spots with an unsatifactory spot morphology and large spot-to-spot variations. Spots might deviate from the per- fect round shape especially when a contact printer is used, and more signifi- cantly, the deposited compounds often display a nonuniform distribution within the spots. Imperfect spots might introduce a large experimental error that com- promises the absolute accuracy of the individual measurement.
- 41. Quality Considerations and Selection of Surface Chemistry 19 Fig. 1. Slide quality test and effect of deposits on the slide surface on spot mor- phology. (A,B) Laser scan images (ScanArray5000, Perkin Elmer, Downers Grove, IL; details 17 × 4.3 mm2) of commercial slides obtained from different manufacturers. Excitation of fluorescence at 543 nm (laser intensity 100%) and detection at 570 nm (photomultiplier tube setting 100%) shows significant differences in the quality of the slide coatings. In this comparison, only slide (A) should be considered for the produc- tion of a microarray. Structures on slide (B) are presumably caused by drying effects during slide production. Inserts show a line profile of the marked region. (C) Deposits on the slide surface result in bad spot quality as illustrated for spots of a Cy3-labeled 13-mer oligonucleotide on a commercial epoxy silane slide of insufficient quality (scan obtained with LS400, Tecan, Salzburg, Austria).
- 42. The sensitivity of spot formation to surface artifacts can be used for a simple slide quality test and can serve to assess the overall quality of whole batches of slides from manufacturers, as according to our experience, a single slide most often represents a batch of 10–25 slides. Using the advantage of piezo printers that are able to deliver pL drops in a highly accurate and reproducible manner, printing a dye-labeled oligonucleotide in some thousand replicates results in an array of spots showing—in an ideal case—identical fluorescence intensities and spot morphology (Fig. 2A). Deviations from a uniform spot intensity across the whole slide surface such as gradients (large-range artifacts, Fig. 2C) or deformed spots (small-range imperfections, Fig. 2B) suggest a bad overall slide quality (see Notes 3 and 4). 2.2. Overview of Slide Surface Chemistry and Immobilization Procedures in the Production of Microarrays 2.2.1. General Considerations on Surface Chemistry and Immobilization Procedures The concept of surface chemistry is directly related to the immobilization of probe molecules. A precondition for the production of microarrays is a surface with a suitable chemical coating. Uncoated clean glass cannot be used because it is too hydrophilic and strongly adsorbs components from the air that change the surface in an uncontrolled way. Moreover, because of lacking binding function- alities, a specific immobilization of the probe molecules is impossible. The large spectrum of surface chemistries used for microarray applications can be repre- sented by their simplified molecular architecture as shown in Fig. 3. The most simple chemical coatings consist of a layer of silane with a reactive terminal group (Fig. 3A). Additionally, spacer groups that can be introduced by the reac- tion with a bifunctional crosslinker increase the distance from the glass surface and ensure a more solution-like behaviour of the binding reaction between probe and target molecules (Fig. 3B). More sophisticated coatings consist of a layer of different types of polymers (Fig. 3C) or dendrimers (Fig. 3D). In all cases, the coating on the slide surface determines the properties of hydrophilicity, long- term chemical stability, binding properties, such as loading capacity and chemi- cal reactivity, the degree of adsorption, and spot size and shape. In addition, the surface chemistry provides the chemical environment for the sample compound and influences stability and accessibility of the immobilized probe molecules. In respect to the latter, there are three general methods for immobilization: the for- mation of strong covalent bonds (1) by a thermal chemical reaction, and (2) ultra- violet (UV) crosslinking, and (3) physical adsorption. It is obvious that the immobilization of different types of probe molecules require a matching surface chemistry in order to obtain optimal experimental results. Immobilization condi- tions are extensively discussed in the subsequent Chapter 3. 20 Sobek et al.
- 43. Quality Considerations and Selection of Surface Chemistry 21 Fig. 2. Line scan through a number of replicate spots. (A) Uniform spots on a high- quality slide surface. The standard deviation of fluorescence intensity is 5% (40 spots). (B) Because of deposits on the slide surface spot shape and intensity is strongly dis- torted in the middle part of the scan. (C) Inhomogeneous coating results in a strong gra- dient in spot intensity. (For verification, the slide was turned by 180° and scanned again to exclude effects arising from scanning out of focal plane.)
- 44. The cost of the slides might increase from simple silane slides to linker- modified and then more from polymer to dendrimer slides. The following para- graphs provide recommendations on the selection of suitable surfaces for various applications (1). 2.2.2. Oligonucleotide Microarrays Now-a-days oligonucleotide arrays can be considered a standard application featuring a number of advantages over cDNA arrays. Because oligonucleotides modified with a nucleophilic linker react under mild conditions with elec- trophilic groups including glycidyloxy (epoxy), aldehyde, isothiocyanate, and activated ester (e.g., N-hydroxysuccinimide [NHS]), slides presenting these functional groups are preferably used. Aminosilane-coated slides does not pro- vide a suitable functionality to covalently immobilize oligonucleotides in a mild and specific manner. On these slides, immobilization can only be achieved by applying harsh conditions, such as heating to 80°C or UV crosslinking (2). In many articles, epoxysilane- and aldehyde-modified slides (prepared by the reaction of aminosilane slides with glutaraldehyde) are successfully used for oligonucleotide applications (refer to publications). However, when comparing processing protocols for epoxysilane- and aldehyde-modified slides, the former has clear advantage regarding simplicity, processing time, flexibility with regard to the blocking reagent, no harmful and toxic chemicals (sodium boro- hydride and sensitive) and hence less cost for waste disposal. It has been found that there is no need for a (rather expensive and sensitive) nucleophilic linker group to immobilize long oligonucleotides at surfaces presenting epoxy groups (see Chapter 3). 22 Sobek et al. Fig. 3. Schematic representation of slide surface architecture. (A) Simple silane- based slides. (B) Linker-modified slides. (C) Polymer-coated slides. (D) Dendrimer- coated slides. The representation is simplified such that no structural effects are taken into account (molecular orientational effects, silane multilayer formation, cross-linking of polymers, and gel formation). Black spheres are linking groups introduced by the chemical synthesis of the slide. Gray spheres are reactive groups. On polymer slides of type 3C these groups might be either reactive functional groups or nonreactive groups, for example, amide. Structures are not drawn to scale.
- 45. To achieve functional immobilization of oligonucleotides smaller than about 20 nt requires a more careful selection of a matching surface chemistry than for com- monly used longer oligonucleotides. Becuase of the short length of the molecules, the accessibility of the immobilized probe by the target molecule can be severely hin- dered (3), longer reaction times are required when hybridized with large target mol- ecules such as cDNA. This is caused by sterical hindrances and by the particular conditions in close proximity to surfaces, where diffusion rate constants are signifi- cantly reduced (4). Moreover, surface charges change the properties and reactivity of the immobilized molecules, leading to substantial differences in hybridization ther- modynamics (5). In order to avoid these effects and to accelerate binding reactions with a target molecule, slides of type 3B–D (according to Fig. 3) are required to ensure a more solution-like interaction of probe and target molecules. 2.2.3. cDNA Microarrays DNA of high-molecular weight, such as cDNA or bacterial artificial chromo- somes (BACs) are usually immobilized on two-dimensional (2D) slide surfaces of type 3A (according to Fig. 3) (6). BACs consist of a mixture of different DNA molecules of different size that show heterogeneous properties on a slide surface regarding the spot morphology. Traditionally, aminosilane- and epoxysilane-coated slides are used for these applications. Comparing the two types of surfaces, aminosilane-coated slides offer some advantages with respect to flexibility in the choice of spotting solutions and the quality of the resulting spot morphology. Frequently used spotting solutions, such as dimethyl sulfox- ide (DMSO) and a saline-citrate buffer (3X SSC) can serve as examples. DMSO is a very useful solvent for many different organic molecules and a 50% aqueous solution is often used for spotting DNA. This solution denatures DNA so that single strands are deposited and immobilized. Thereby, no protocol step for strand separation on the slide surface is necessary and spares the slides to be boiled at 95°C, which is a harsh treatment (see experiments and Fig. 1 in Chapter 3). In addition, spotting cDNA in 50% DMSO to aminosilane-coated slides results in a good spot morphology, which is not the case for epoxysilane- coated slides that are incompatible with DMSO in water and produce spots of undefined shape and morphology. In many cases, 3X SSC is a very good spotting buffer in combination with sur- faces coated with epoxysilane or aminosilane and results in spots of excellent morphology for many types of probe molecules. However, in the case of BACs printed in 3X SSC the spot morphology on epoxysilane slides is unsat- isfactory. The same solution printed to aminosilane slides results in spots of a very good morphology, which illustrates the sensitvity of the spot morphology to the combined properties of the spotting solution and the surface coating. As out- lined next, spotting dye-labeled model compounds in recommended solutions Quality Considerations and Selection of Surface Chemistry 23
- 46. to the slide of choice shows the resulting spot morphology right away (see Chapter 3, Subheadings 2.1.2. and 2.1.4.). This helps to choose a functional com- bination of spotting solution and slide surface without the need of hybridization. 2.2.4. Peptide Microarrays For applications that require the immobilization of molecules of low-molecular weight linker-modified slides of type 3B, polymer slides of type 3C, and den- drimer slides of type 3D (according to Fig. 3) should be used for the reasons men- tioned earlier (see Subheading 2.2.2.). If the probe molecules contain a sufficiently long linker, simple silane-based slides of type 3A can be used. In any case, a matching surface chemistry is indispensable. There are many examples published, offering a variety of sophisticated immobilization strategies (7–14). In most cases, home-made slide surfaces were used including linker-modified aminosilane (12,15–17) and epoxysilane slides (12), epoxy-activated polyethyl- ene glycol (PEG)-coated surfaces (18), aldehyde-modified slides (11,19,20), semicarbazide slides (13,21,22), and alkanethiolates forming self-assembled monolayers on gold (9,10,23), among others. Our laboratory have successfully used in-house made PEG-coated slides for spotting peptide arrays for epitope mapping of corresponding antibodies (24). There are only a limited selection of type 3B and 3C slides on the market. As a consequence, many microarray groups prepare slides of their own. 2.2.5. Carbohydrate Microarrays Carbohydrate microarrays are a relatively new application and not yet well established. The biggest problem in producing a carbohydrate microarray is the limited availability of defined carbohydrate samples (25). For carbohydrates of low-molecular mass, the best method for immobilization is covalent (9,26–31) or noncovalent coupling (32–34) using a linker with a specific coupling chem- istry. However, this requires sophisticated chemical modifications as carbohy- drates from natural sources usually lack such a suitable linker needed for a site-directed immobilization to the surface. An alternative is immobilization in the absence of a modification of the probe molecules. Microbial polysaccharides of high-molecular mass (35) and neoglycolipids (36) were adsorbed to nitrocellulose-coated slides. Bryan and Wong immobilized unmodified di- and trisaccharides to nonpolar polystyrene microtiter plates by adsorption (34,37). Because to their high content of nucle- ophilic hydroxyl groups carbohydrates easily immobilize at surfaces presenting electrophilic groups (e.g., epoxy or NHS ester groups). Fluorescein-labeled dextrane was immobilized to epoxysilane of type 3B and other electrophilic- activated polymer surfaces of type 3C (see Fig. 3) in model experiments to determine processing parameter (see Chapter 3). However, without site-specific 24 Sobek et al.
- 47. immobilization chemistry the carbohydrates are bound in a random distribution at the surface. In turn, this undefined orientation might lead to blocking of molecular recognition sites, a situation similar to that observed for protein and antibody microarrays (38–40). Feizi et al. (27) presented an overview of immo- bilization methods for carbohydrates. Slide surface coatings used so far include thioles on gold (28) or glass (31,41), nitrocellulose (35,36,42), and (oxidized black) polystyrene (37,43). 2.2.6. Small Molecule Microarrays The term “small molecule” refers to chemical compounds of low-molecular mass that can be extracted from natural sources or produced synthetically (44–46). For an overview on the use of small molecules in microarray applica- tions see refs. 16,47–51. The molecular structures of these probes can be highly diverse. A large variety of chemical reactions leading to a stable chemical bond and efficiently coupling the probe to the surface may be applied for immobiliza- tion. Usually, in the course of the chemical synthesis of probes, a building block consisting of a linker and a reactive group are introduced for which a slide with matching surface chemistry is available. As discussed for short oligonucleotide, carbohydrate, and peptide microarrays, two main principles must be followed: First, a site-directed immobilization in terms of matching chemical groups for coupling the probe to the surface must be applied. Second, a suitable linker to gain distance from the surface is needed to obtain a good accessibility of the probes by the target molecules. Examples for immobilization of small molecules include the extensive work of the Schreiber group coupling probes containing a hydroxyl group to a chlorinated surface (52–55), and the reaction of azide groups with a phosphane-coated sur- face in a Staudinger ligation reaction (56). Aminosilane-coated slides were mod- ified with a bifunctional crosslinker including maleimide derivatives (15), a PEG derivative (18), or a diazobenzylidene (57). Hoff et al. (15) used a chemolabile linker for a slow release of immobilized probes for uptake by cells incubated on the microarray. An alternative surface chemistry applied to small molecule microarrays includes photoaffinity labeling of the surface with a light sensitive 3H-diazirinyl presented by Kanoh et al. (58). At UV irradiation, this chemical group decomposes and forms a reactive electrophilic carbene that efficiently reacts with nucleophilic groups in steroids. The authors state that because of the high reactivity of the carbene, structurally distinct molecules can be immobilized. A completely different approach for the production and processing of small molecule microarrays was invented by Gosalia and Diamond (59). Their probes were dissolved in a nonvolatile glycerol/DMSO mixture and were spotted to cleaned blank glass slides. These nanoliter droplets did not evaporate and were used as stable liquid microreactors. Without the need for immobilization and Quality Considerations and Selection of Surface Chemistry 25
- 48. washing procedures, target compounds were subsequently applied by aerosol dep- osition. This method allows for the transfer of the 96-well plate assay format to a microarray while requiring much smaller volumes for a screen in liquid solution. Note that some authors use the term small molecule microarray in the con- text of hybridization of small molecule-tag conjugates to, for example, an oligonucleotide array (60). In this type of application, PNA or oligonucleotides (61) are used as decoding tag. 2.2.7. Antibody Microarrays Unlike the examples described earlier, antibodies and other proteins are large molecules that immobilize at nearly all surfaces by passive adsorption. In addi- tion, covalent bonds are formed if the slide surface provides reactive electrophilic groups. Most importantly, as the biological function of proteins depends on their three-dimensional structure, conformational changes must be avoided. In practice, this is rarely possible to the full extent and explains why certain proteins immo- bilized at surfaces do not show the expected function observed in solution (62,63). The degree of denaturation of a protein on a surface strongly depends on the chemical nature of the coating. Although many surfaces are prone to denature proteins, PEG-coated surfaces and hydrogels are used to reduce the risk of protein denaturation (64,65). Immobilization of antibodies on 2D surfaces was optimized by Peluso et al. (66). Angenendt et al. (67) described an overview on methods of specific immobiliza- tion of antibodies and proteins. Some commercial antibody and protein microar- rays are based on nitrocellulose membrane slides. Unfortunately, application of these slides is strongly limited owing to the generation of a large background sig- nal upon laser excitation of fluorophores caused by extensive stray light arising from the some 10-μm thick nitrocellulose membrane (68). A variety of phosphorylation specific antibodies have been successfully used on all types of slides recommended earlier, which illustrates that antibodies are more stable than many other proteins and that a variety of surfaces can be used for antibody applications (69). 3. Notes 1. Never touch the surface of a microarray or wipe off dust and other particles. Deposits should be removed exclusively by clean compressed air or inert gases. 2. Coated but unprocessed slides are sensitive to air (amino groups) and humidity (nearly all reactive groups used on slides). Keep in mind that there is only a mono- layer of molecules with reactive groups immobilized at a 2D slide surface. Once a package is opened, the slides should be used immediately or sealed appropri- ately. The best way to store slides is under nitrogen in a desiccated box. Slides can usually be used past the manufacturer recommended date if properly stored. 26 Sobek et al.
- 49. Some reactive groups (NHS ester-activated slides) degrade even when stored under optimal condition but can be reactivated (70). 3. Spotted slides should be stored without further processing (as long as there is no specific reason for processing, for example, as a result of a change in spot mor- phology with time caused by properties of the spotting solution). There is a large amount of probe in the spot covering and thereby protecting those molecules immobilized at the surface. An exception are slide coatings based on nitrocellu- lose and nylon, where the three-dimensional matrix protects the spotted probe. 4. Processed slides stored desiccated under nitrogen in the dark can be used for months for later scanning. Experiments performed during the summer (2005) have shown that dyes degrade on the surface of microarrays upon exposed to air even when stored under exclusion of light. Results of dye stability tests clearly corre- late with the air ozone level (71). Acknowledgment JS likes to thank Prof. Dr. Joe Jiricny (University of Zurich, ETH Zurich), Dr. Orlando Schärer (Stony Brook University Hospital), and Dr. Philip Day (University of Manchester) for their extensive help getting started with slide development, microarray production, and hybridization experiments. References 1. For availability of microarray slides, see Sobek, J. and Schlapbach, R. (2004) Substrate architecture and functionality define the properties and performance of DNA, peptide, protein, and carbohydrate microarrays. Pharmagenomics Sept.15, 32–44. 2. Wang, H. -Y., Malek, R. L., Kwitek, A. E., et al. (2003) Assessing unmodified 70-mer oligonucleotide probe performance on glass-slide microarrays. Genome Biol. 4, R5. 3. Butler, J. E. (2000) Solid supports in enzyme-linked immunosorbent assay and other solid-phase immunoassays. Methods 22, 4–23. 4. Chan, V., Graves, D. J., Fortina, P., and McKenzie, S. E. (1997) Adsorption and surface diffusion of DNA oligonucleotides at liquid/solid interfaces. Langmuir 13, 320–329. 5. Vainrub, A. and Pettitt, B. M. (2003) Surface electrostatic effects in oligonu- cleotide microarrays: control and optimization of binding thermodynamics. Biopolymers 68, 265–270. 6. Taylor, S., Smith, S., Windle, B., and Guiseppi-Elie, A. (2003) Impact of surface chemistry and blocking strategies on DNA microarrays. Nucleic Acids Res. 31, E87. 7. Yeo, D. S.Y., Srinivasan, R., Chen, G.Y. J., andYao, S. Q. (2004) Expanded utility of the native chemical ligation reaction. Chem. Eur. J. 10, 4664–4672. 8. Melnyk, O., Duburcq, X., Olivier, C., Urbès, F., Auriault, C., and Gras-Masse, H. (2002) Peptide arrays for highly sensitive and specific antibody-binding fluores- cence assays. Bioconjugate Chem. 13, 713–720. Quality Considerations and Selection of Surface Chemistry 27
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- 52. Other documents randomly have different content
- 56. The Project Gutenberg eBook of Annali d'Italia, vol. 2
- 57. This ebook is for the use of anyone anywhere in the United States and most other parts of the world at no cost and with almost no restrictions whatsoever. You may copy it, give it away or re-use it under the terms of the Project Gutenberg License included with this ebook or online at www.gutenberg.org. If you are not located in the United States, you will have to check the laws of the country where you are located before using this eBook. Title: Annali d'Italia, vol. 2 Author: Lodovico Antonio Muratori Release date: August 27, 2013 [eBook #43575] Most recently updated: October 23, 2024 Language: Italian Credits: Produced by Carlo Traverso, Claudio Paganelli, Barbara Magni and the Online Distributed Proofreading Team at http://www.pgdp.net (This file was produced from images generously made available by The Internet Archive) *** START OF THE PROJECT GUTENBERG EBOOK ANNALI D'ITALIA, VOL. 2 ***
- 59. ANNALI D ' I TA L I A 2
- 60. ANNALI D ' I TA L I A DAL PRINCIPIO DELL'ERA VOLGARE SINO ALL'ANNO 1750 COMPILATI D A L . A N T O N I O M U R A T O R I E CONTINUATI SINO A' GIORNI NOSTRI Quinta Edizione Veneta VOLUME SECONDO V E N E Z I A DAL PREMIATO STAB. DI G. ANTONELLI ED. 1844
- 61. INDICE
- 62. A N N A L I D ' I TA L I A DAL PRINCIPIO DELL'ERA VOLGARE FINO ALL'ANNO 1500
- 63. Anno di Cristo CCCXLI. Indizione XIV. Giulio papa 5. Costanzo e Costante imperadori 5. Consoli Antonio Marcellino e Petronio Probino. Un'iscrizione che si legge nella mia Raccolta [Thes. Novus Inscript., pag. 377.], quando pur sia indubitata reliquia dell'antichità, ci assicura dei nomi di questi consoli, in addietro ignoti. Aurelio Celsino dal dì 25 di febbraio cominciò ad esercitare la prefettura di Roma. Sul fine di giugno diede Costanzo Augusto una legge in Lauriaco [L. 31, de Decurion., Cod. Theodos.], creduto dal Gotofredo luogo della Batavia, ma che più verisimilmente fu il Lauriaco, luogo insigne e colonia de' Romani, posta alle parti superiori del Danubio. Era questo principe divenuto signor delle Gallie, e colà dovette accorrere [Idacius, in Fastis.], perchè i Franchi, passato il Reno, metteano a sacco le vicine contrade romane. Abbiamo da san Girolamo [Hieron., in Chron.] che seguirono fra que' Barbari e le armate di Costante varii combattimenti, ma senza dichiararsi la fortuna per alcuna delle parti. Libanio [Liban., Orat. III.], descrivendo a lungo i costumi e il genio de' Franchi d'allora, li dipinge per gente turbolenta ed inquieta, a cui il
- 64. riposo riusciva un supplizio. Solamente nell'anno seguente ebbe fine questa guerra. Tanto il medesimo san Girolamo che Idacio mettono sotto il presente anno spaventosi tremuoti che fecero traballare moltissime città dell'Oriente. Tennero in quest'anno gli ariani un conciliabolo in Antiochia, per alterare i decreti sacrosanti del concilio niceno. Appena terminata fu la sacrilega loro assemblea, che il tremuoto cominciò a scuotere orribilmente la misera città, siccome attestano Socrate [Socrates, Histor., lib. 2, cap. 11.] e Sozomeno [Sozomenus, Histor., lib. 3, cap. 6.], e quasi per un anno si andarono sentendo varie altre scosse. Non parla Teofane [Theophanes, in Chronogr.] se non di tre giorni, ne' quali probabilmente quella città fu in maggior pericolo. Lo stesso autore nota che circa questi tempi Costanzo Augusto cinse di forti mura e fortificò in altre guise Amida, città della Mesopotamia, situata presso il fiume Tigri, acciocchè servisse di antemurale contro ai Persiani. Ammiano [Ammianus, Histor., lib. 18, cap. 9.], scrittore di maggior credito, all'incontro, scrive che molto prima d'ora, cioè vivente ancora il padre, Costanzo Cesare con torri e mura fece divenir quel luogo un'importante fortezza, di cui sempre più crebbe la popolazione e la fama ne' tempi susseguenti. Durava tuttavia la guerra coi Persiani, ovvero, se Socrate [Socrat., Histor., lib. 2, cap. 25.] non s'inganna, essa ebbe principio in questi medesimi tempi; ma quali azioni militari si facessero, non è pervenuto a nostra notizia. Già abbiam detto che Costantino il Grande con varii editti e in altre guise si studiò di abolir le superstizioni del paganesimo, distrusse moltissimi templi de' gentili, vietò gli empii loro sagrifizii: il che vien confermato da Socrate [Idem, ibid., lib. 1, cap. 8.], da Teodoreto [Theodoret., in Histor. Eccl.], da Teofane [Theoph., Chronogr.] e da altri. Ma lo svellere dal cuore di tanta gente gli antichi errori e riti, difficil cosa riusciva nella pratica. Costante Augusto nell'anno presente, siccome principe di massime cattoliche e di zelo cristiano, per eseguire eziandio ciò che il padre gli avea premurosamente raccomandato, pubblicò una legge, con cui, confermando gli editti paterni [L. 2, de Paganis., Cod. Theod.], sotto rigorose pene abolisce i sagrifizii de' pagani, e per conseguenza ancora il culto degl'idoli. Siffatti editti, e l'esempio de' principi seguaci della legge di Cristo, furono quegli arieti che diedero un gran tracollo
- 65. al gentilesimo, con ridurlo a poco a poco all'ultima rovina. Ma se ad occhio veniva meno la falsa religion de' pagani, per cura massimamente dell'Augusto Costante, andavano ben crescendo in questi tempi le forze dell'arianismo in Oriente con discapito della Chiesa cattolica, per la protezion che avea preso di quella fazione l'Augusto Costanzo. Le insigni sedie episcopali di Alessandria, Antiochia e Costantinopoli vennero in questi tempi occupate da' vescovi ariani [Socrat., lib. 5, cap. 7. Theoph. Cedr.]: e tutte le chiese d'essa città di Costantinopoli caddero in poter de' medesimi eretici. Ma intorno a ciò è da consultare la storia ecclesiastica. Grande solennità nel presente anno fu fatta in Antiochia per la dedicazione di questa magnifica cattedrale, cominciata da Costantino il Grande, e compiuta solamente ora per cura del suddetto imperadore Costanzo.
- 66. Anno di Cristo CCCXLII. Indizione XV. Giulio papa 6. Costanzo e Costante imperadori 6. Consoli Flavio Giulio Costanzo Augusto per la terza volta e Flavio Giulio Costante Augusto per la seconda. Ad Aurelio Celsino nella prefettura di Roma succedette in quest'anno nelle calende d'aprile Mavorzio Lolliano [ Cuspinianus. Panvinius. Bucherius.], il cui impiego durò sino al dì 14 di luglio, con avere per successore Acone (ossia Aconio) Catulino (ossia Catullino) Filomazio (o pur Filoniano). All'anno presente riferisce il Gotofredo [ Gothofred., Chron. Cod. Theodos.] un editto [ L. 3, de Paganis, Cod. eod. Theod.] di Costante Augusto, dato nel dì primo di novembre, e indirizzato al medesimo Catullino prefetto di Roma, in cui ordina che, quantunque s'abbia da abolire affatto la superstizione pagana, pure non si demoliscano i templi situati fuori di Roma, per non levare al popolo romano i divertimenti dei giuochi circensi e combattimenti che aveano presa la origine da que' medesimi templi. Nè già paresse per questo raffreddato punto lo zelo di questo principe in favore del cristianesimo, perchè egli non altro volle che conservar le mura e le
- 67. fabbriche materiali di que' templi, ma con obbligo di sbarbicar tutto quel che sapeva di superstizione gentilesca, come idoli, altari e sagrifizii. Fors'anche non dispiaceva ad alcuni accorti cristiani che restassero in piedi que' superbi edifizii, per convertirli un dì in onore del vero Dio. Ma che in tanti altri luoghi venissero abbattuti i templi de' gentili, Giulio Firmico [Julius Firmicus, de error. prof. Rel.], che circa questi tempi fioriva e scrisse i suoi libri, ce ne assicura. Fino al presente anno sostennero i Franchi la guerra nelle Gallie contra dell'Augusto Costante [ Hieronymus, in Chron. Idacius, in Fastis. Socrates, lib. 2, cap. 13. Theoph., in Chron.]. Tali percosse nondimeno dovettero riportare dall'armi romane, che finalmente si ridussero a chiedere pace. Un trattato di amicizia e lega conchiuso con Costante li fece ripassare il Reno. Libanio [ Liban., Orat. III.] con oratoria magniloquenza lasciò scritto che il solo terrore del nome di Costante obbligò que' popoli barbari ad implorare un accordo, senza dire che fossero domati coll'armi, come scrissero tanti altri. Aggiugne ch'essi Franchi riceverono dalla mano di Costante i loro principi, e stettero poi quieti per qualche tempo. Occorse nell'anno presente in Costantinopoli più d'una sedizione fra i cattolici ed ariani [ Socrates, lib. 2, cap. 13. Sozomenus, Hist. Eccl. Idacius, in Fastis. Hieronym., in Chron.], da che Costanzo Augusto, sposata affatto la fazione degli ultimi, mandò ordine che fosse da quella cattedra cacciato Paolo vescovo cattolico, per introdurvi Macedonio ariano. Crebbe un dì a tal segno l'impazienza e il furor della plebe cattolica, che andarono ad incendiar la casa di Ermogene generale dell'armi, a cui era venuto l'ordine dell'imperadore di eseguir la deposizione del vescovo cattolico; e messe le mani addosso al medesimo Ermogene, lo strascinarono per la città, e lo uccisero. Costanzo, che allora si trovava ad Antiochia, udita cotal novità, tosto per le poste volò a Costantinopoli: cacciò Paolo e gastigò il popolo, con privarlo della metà del grano, che per istituzione di Costantino gli era somministrato gratis ogni anno; cioè di ottanta mila moggia o misure ridusse il dono a sole quaranta mila.
- 68. Anno di Cristo CCCXLIII. Indizione I. Giulio papa 7. Costanzo e Costante imperadori 7. Consoli Marco Mecio Memmio Furio Baburio Ceciliano Procolo e Romolo. Questa gran filza di cognomi data al primo console, cioè a Procolo, si truova in un'iscrizione creduta spettante a lui, e rapportata dal Panvinio e Grutero. Non Balburio, come essi hanno, ma Baburio viene appellato nelle schede di Ciriaco, che riferisce lo stesso marmo. Il secondo console dal suddetto Panvinio, che cita un'iscrizione, vien chiamato Flavio Pisidio Romolo. Vopisco, nella Vita d'Aureliano [Vopiscus, in Aurel.], ci rappresenta questo Procolo per uomo abbondante, non so se più di ricchezze o di vanità, scrivendo essersi poco fa veduto il consolato di Furio Procolo solennizzato con tale sfoggio nel circo, che non già premii, ma patrimonii interi parve che fossero donati ai vincitori nella corsa de' cavalli. Ci fan conoscere tali parole in che tempo Vopisco fiorisse e scrivesse. Nella prefettura di Roma continuò ancora per quest'anno Aconio Catullino. Dappoichè la pace stabilita coi Franchi rimise la calma in tutte le Gallie, Costante Augusto, il quale si truovava in Bologna di Picardia nel
- 69. gennaio dell'anno presente [Gothofred., Chron. Cod. Theodos.], volle farsi vedere anche ai popoli della Bretagna, e passò nel furore del verno colà con tutta felicità. Se prestiam fede a Libanio [Liban., Orat. III.], guerra non v'era che il chiamasse di là dal mare, ma solo timor di guerra; e da Ammiano Marcellino [Ammianus, lib. 20, cap. 1.] si ha abbastanza per credere che i Barbari di quella grand'isola avessero fatta almen qualche scorreria nel paese de' Romani. Per altro, che non succedessero battaglie e vittorie in quelle parti, si può argomentare dal suddetto Libanio, giacchè egli di niuna fa menzione. Truovansi nulladimeno alcune medaglie, dove egli è appellato [Mediob., in Numismat. Imperator.] debellatore e trionfatore delle nazioni barbare, le quali, se non sono parti della sola bugiarda adulazione, possono indicare qualche vantaggio delle sue armi in quelle contrade ancora. Oltre di che, Giulio Firmico [Julius Firmicus, de error. profan. Rel.], parlando ai due Augusti, dice, che dopo aver essi abbattuti i templi de' gentili nell'anno 341, Dio avea prosperate le lor armi; che aveano vinti i nemici, dilatato l'imperio; che i Britanni, all'improvviso comparir dell'imperadore, s'erano intimoriti. Truovasi poi esso Augusto nel dì 30 di giugno ritornato a Treveri, dove è data una sua legge. Ci fanno poi altre leggi vedere Costanzo Augusto in Antiochia, in Cizico, in Jerapoli, tutte città dell'Asia, imperocchè non gli lasciava godere riposo la guerra sempre viva coi Persiani. Osserviamo ancora in una delle sue leggi [L. 35, de Decur., Cod. Theod.] ch'egli chiamò a militare in quest'anno i figliuoli dei veterani, purchè giunti all'età di sedici anni, per bisogno certamente di quella guerra. Non so io dire quale credenza si meriti Teofane [Theoph., in Chronogr.], allorchè scrive che circa questi tempi Costanzo, dopo aver vinti gli Assirii, cioè i Persiani suddetti, trionfò. Niuno de' più antichi e vicini storici a lui attribuisce alcuna memorabil vittoria di que' popoli, e molto meno un vero trionfo. Abbiamo inoltre dal medesimo Teofane che la città di Salamina nell'isola di Cipri per un fierissimo tremuoto restò la maggior parte smantellata; siccome ancora circa questi tempi ebbe principio la persecuzione mossa da Sapore re di Persia contra de' cristiani abitanti ne' paesi di suo dominio.
- 70. Anno di Cristo CCCXLIV. Indizione II. Giulio papa 8. Costanzo e Costante imperadori 8. Consoli Leonzio e Sallustio. Nel dì 11 d'aprile ad Acone, ossia Aconio, Catullino succedette nella prefettura di Roma Quinto Rustico. Nulla di considerabile ci somministra per questo anno la storia, se non che truoviamo una legge [L. 3, de excusat. artific.], con cui Costanzo Augusto concede delle esenzioni ai professori di meccanica, architettura e ai livellatori delle acque. Il genio edificatorio veramente non mancò a questo imperadore, ed egli lasciò molte suntuose fabbriche da lui fatte in Costantinopoli, Antiochia ed altri luoghi. Ma se egli coll'una mano innalzava materiali edifizii nel suo dominio, coll'altra incautamente si studiava di atterrare e distruggere la dottrina e Chiesa cattolica, lasciandosi aggirare a lor talento dai seguaci dello eresiarca Ario. Però in questi tempi smisuratamente prevalse in Oriente la lor fazione: laddove Costante Augusto in Occidente, con dichiararsi protettore dei dogmi del concilio niceno, divenne scudo della Chiesa cattolica. Se in Oriente si tenevano conciliaboli contra la fede nicena,
- 71. in Occidente ancora si formavano concilii per sostenerla. Ma intorno a ciò mi rimetto alla storia ecclesiastica. Intanto era flagellato da Dio l'imperador Costanzo col tarlo della guerra persiana; e benchè Teofane [Theoph., in Chronogr.] ancora sotto quest'anno racconti che vennero alle mani le due armate romana e persiana, e che gran numero di que' Barbari lasciò la vita sul campo; pure, poco o nulla servirono questi pretesi vantaggi, perchè più che mai vigorosi i Persiani continuarono a fare il ballo sulle terre romane, senza che mai riuscisse ai Romani di cavalcare sul paese nemico. Abbiamo poi da san Girolamo [Hieronymus, in Chronico.] e dal suddetto Teofane che nell'anno presente Neocesarea, città la più riguardevol del Ponto, fu interamente rovesciata a terra da un orrendo tremuoto colla morte della maggior parte del popolo, essendosi solamente salvata la cattedrale fabbricata da san Gregorio Taumaturgo colla casa episcopale, dove esso vescovo e chiunque ivi si trovò rimasero esenti da quello eccidio.
- 72. Anno di Cristo CCCXLV. Indizione III. Giulio papa 9. Costanzo e Costante imperadori 9. Consoli Amanzio ed Albino. Secondo il Catalogo del Cuspiniano e del Bucherio, nel dì 5 di luglio Probino fu creato prefetto di Roma. Una legge [L. 7, de petition., Cod. Theod.] di Costante Augusto, data nel dì 15 maggio, ci fa vedere questo imperador ritornato dalla Bretagna a Treveri. Però non so se sussista l'aver creduto il Tillemont [Tillemont, Mémoires des Empereurs et de l'Histoire Ecclesiastique.] ch'esso Augusto verso il fine del medesimo mese fosse in Milano, dove invitò lo sbattuto santo Atanasio, per patrocinarlo contra la prepotenza degli ariani. Certamente cominciò verso questi tempi il cattolico Augusto a tempestar con lettere il fratello Costanzo, acciocchè si tenesse un concilio valevole a metter fine a tante turbolenze della Chiesa. Ma non si arrivò a questo se non nell'anno 347, siccome allora accenneremo. Da una legge del Codice Teodosiano [L. 5, de exactionib., Cod. Theod.] apprendiamo che l'Augusto Costanzo, nel dì 12 di maggio del presente anno, si trovava in Nisibi città della Mesopotamia, e, senza fallo, per accudire alla
- 73. guerra coi Persiani. Abbiamo poi da san Girolamo [Hieron., in Chronico.] e da Teofane [Theoph., in Chronogr.] che in quest'anno ancora i tremuoti cagionarono nuove rovine in varie città. Fra le altre la marittima di Epidamno ossia di Durazzo, città della Dalmazia, restò quasi affatto abissata. Anche in Roma per tre giorni sì gagliarde furono le scosse, che si paventò l'universal caduta delle fabbriche. Nella Campania dodici città andarono per terra; e l'isola, o, vogliam dire, la città di Rodi, fieramente anch'essa risentì la medesima sciagura. Se crediamo alla Cronica Alessandrina [Chronic. Alexandrinum.], Costanzo Augusto cominciò in quest'anno la fabbrica delle sue terme in Costantinopoli; ma intorno a ciò è da vedere il Du-Cange [Du-Cange, Hist. Byz.], che rapporta altre notizie spettanti a quell'insigne edifizio.
- 74. Anno di Cristo CCCXLVI. Indizione IV. Giulio papa 10. Costanzo e Costante imperadori 10. Consoli Flavio Giulio Costanzo Augusto per la quarta volta e Flavio Giulio Costante Augusto per la terza. Perchè non si dovettero speditamente accordare i due Augusti intorno al prendere insieme il consolato, o pure a notificarlo, noi troviamo che nel Catalogo del Bucherio e in un concilio di Colonia per li primi mesi dell'anno presente non si contavano i consoli nuovi; perciò l'anno veniva indicato colla formola di dopo il consolato di Amanzio ed Albino. Nella prefettura di Roma stette Probino sino al dì 26 di dicembre dell'anno presente [Cuspinianus. Panvinius. Bucherius.], ed allora in quella carica succedette Placido. Noi ricaviamo dalle leggi del Codice Teodosiano [Gothofred., Chronolog. Cod. Theodos.] spettanti a quest'anno che Costante Augusto era in Cesena nel dì 23 di maggio, e in Milano nel dì 21 di giugno. Dall'Italia dovette egli passare in Macedonia, perchè abbiamo una legge di lui data in Tessalonica nel dì 6 di dicembre. Per conto dell'Augusto Costanzo, egli non altrove comparisce che in Costantinopoli, dove confermò o pur concedette
- 75. molte esenzioni agli ecclesiastici. All'anno presente riferisce san Girolamo [Hieron., in Chron.] la fabbrica del porto di Seleucia, città famosa della Soria, poche miglia distante da Antiochia, capitale dell'Oriente. Anche Giuliano [Julian., Orat. I.] e Libanio [Liban., Orat. III.] parlano di questa impresa, che riuscì d'incredibile spesa al pubblico, perchè per formare quel porto non già alla sboccatura del fiume Oronte, come talun suppone, ma bensì alla stessa Seleucia, convenne tagliar molti scogli e un pezzo di montagna, che impedivano l'accesso alle navi, e rendevano pericolosa e poco utile una specie di porto che quivi anche antecedentemente era. Perchè la corte dell'imperador Costanzo per lo più soggiornava in Antiochia, di incredibil comodo e ricchezza riuscì dipoi a quella città il vicino porto di Seleucia. Teofane [Theophanes, Chronogr.] aggiugne che Costanzo con altre fabbriche ampliò ed adornò la stessa città di Seleucia; ed inoltre abbellì la città di Antarado nella Fenicia, la quale prese allora il nome di Costanza. Mentre poi esso Augusto Costanzo impiegava in questa maniera i suoi pensieri e i tesori, cavati dalle viscere dei sudditi, dietro alle fabbriche, il re di Persia Sapore non lasciava in ozio la forza delle sue armi; e però, secondochè scrive il suddetto Teofane, nell'anno presente si portò per la seconda volta all'assedio della città di Nisibi nella Mesopotamia. Vi stette sotto settantotto giorni, e, non ostante tutti i suoi sforzi, fu in fine obbligato a vergognosamente levare il campo e ritirarsi. Nella Cronica di san Girolamo un tale assedio vien riferito all'anno seguente. Ma cotanto hanno gli antichi moltiplicato il numero degli assedii di Nisibi con discordia fra loro, che non si sa che credere. Verisimilmente un solo assedio fin qui fu fatto, cioè se sussiste il già accennato all'anno 338, un altro non sarà da aggiugnere all'anno presente. Parleremo, andando innanzi, d'altri assedii di quella città. Pare che in quest'anno accadesse una sedizione in Costantinopoli, per cui quel governatore Alessandro restò ferito, e se ne fuggì ad Eraclea. Tornossene ben egli fra poco al suo impiego, ma poco stette ad esser deposto da Costanzo, con succedergli in quel governo Limenio. Libanio [Liban., in ejus vita.] quegli è che ci ha conservata questa notizia, e che sparla forte d'esso Limenio, perchè il buon sofista fu cacciato da Costantinopoli d'ordine suo.
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