![Nylon cDNA Expression Arrays 13
2. Tissue homogenizer (e.g., Polytron or equivalent). For <200 mg of tissue, use a
6-mm probe. For >200 mg of tissue, use a 10-mm probe.
3. [α-32P]dATP (10 µCi/µL; 3000 Ci/mmol) (cat. no. PB10204; Amersham) or
[α-33P]dATP (10 µCi/µL; >2500 Ci/mmol) (cat. no. BF1001; Amersham). Do
not use Amersham’s Redivue or any other dye-containing isotope.
4. Deionized H2O (Milli-Q filtered or equivalent; do not use diethylpyrocarbonate-
treated H2O).
5. Magnetic particle separator (cat. no. Z5331; Promega, Madison, WI). It is
important that you use a separator designed for 0.5-mL tubes.
6. Polypropylene centrifuge tubes: 1.5-mL (cat. no. 72-690-051; Sarstedt), 2-mL
(cat. no. 16-8105-75; PGC), 15-mL (tubes cat. no. 05-562-10D, caps cat. no.
05-562-11E; Fisher), and 50-mL (tubes with caps cat. no. 05-529-1D; Fisher).
Fifteen- and 50-mL tubes should be sterilized with 1% sodium dodecyl sulfate
(SDS) and ethanol before use.
7. 10X dNTP mix (for dATP label; 5 mM each of dCTP, dGTP, dTTP).
8. 10X Random primer mix (N-15) or gene-specific primer mix (see Subhead-
ing 3.4.3.).
9. BD PowerScript™ Reverse Transcriptase and 5X BD PowerScript™ Reaction
Buffer (available exclusively from BD Biosciences Clontech; cat. no. 8460-1).
10. Dithiothreitol (DTT) (100 mM).
11. NucleoSpin® Extraction Kit: NucleoSpin extraction spin columns, 2-mL collec-
tion tubes, buffer NT2, buffer NT3 (add 95% ethanol before use as specified
on the label), buffer NE.
2.3. Reagents for Hybridization, Washing, and Stripping
of Nylon Arrays
1. BD ExpressHyb™ hybridization solution (cat. no. 8015-1).
2. Sheared salmon testes DNA (10 mg/mL) (cat. no. D7656; Sigma).
3. Optional: 10X Denaturing solution (1 M NaOH, 10 mM EDTA) (see Subhead-
ing 3.5.).
4. Optional: 2X Neutralizing solution (1 M NaH2PO4 [pH 7.0]): 27.6 g of
NaH2PO4•H2O). Add 190 mL of H2O, adjust the pH to 7.0 with 10 N NaOH
if necessary, and add H2O to 200 mL. Store at room temperature (see Subhead-
ing 3.5.).
5. Cot-1 DNA (1 mg/mL).
6. 20X saline sodium citrate (SSC), 175.3 g of NaCl, 88.2 g of Na3citrate•2H2O.
Add 900 mL of H2O, adjust the pH to 7.0 with 1 M HCl if necessary, and add
H2O to 1 L. Store at room temperature.
7. 20% SDS: 200 g of SDS. Add H2O to 1 L. Heat to 65°C to dissolve. Store at
room temperature.
8. Wash solution 1: 2X SSC, 1% SDS. Store at room temperature.
9. Wash solution 2: 0.1X SSC, 0.5% SDS. Store at room temperature.](https://image.slidesharecdn.com/18613-250726155514-ac3051e5/85/Functional-Genomics-Methods-and-Protocols-1st-Edition-Charlie-C-Xiang-20-320.jpg)
![[Transcriber's Note: Original text had two Section VIII headings.
Second heading renumbered to IX.]](https://image.slidesharecdn.com/18613-250726155514-ac3051e5/85/Functional-Genomics-Methods-and-Protocols-1st-Edition-Charlie-C-Xiang-53-320.jpg)
Functional Genomics Methods and Protocols 1st Edition Charlie C. Xiang
- 1. Functional Genomics Methods and Protocols 1st Edition Charlie C. Xiang - PDF Download (2025) https://ebookultra.com/download/functional-genomics-methods-and- protocols-1st-edition-charlie-c-xiang/ Visit ebookultra.com today to download the complete set of ebooks or textbooks
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- 5. Functional Genomics Methods and Protocols 1st Edition Charlie C. Xiang Digital Instant Download Author(s): Charlie C. Xiang, Michael J. Brownstein (auth.), Michael J. Brownstein, Arkady B. Khodursky (eds.) ISBN(s): 9781617374746, 1617374741 Edition: 1 File Details: PDF, 5.11 MB Year: 2003 Language: english
- 6. Methods in Molecular BiologyTM Edited by Michael J. Brownstein Arkady B. Khodursky Functional Genomics Methods in Molecular BiologyTM VOLUME 224 Methods and Protocols Edited by Michael J. Brownstein Arkady B. Khodursky Functional Genomics Methods and Protocols
- 7. 1. Fabrication of cDNA Microarrays Xiang, Charlie C.; Brownstein, Michael J. pp. 01-08 2. Nylon cDNA Expression Arrays Jokhadze, George; Chen, Stephen; Granger, Claire; Chenchik, Alex pp. 09-30 3. Plastic Microarrays: A Novel Array Support Combining the Benefi ts of Macro- and Microarrays Munishkin, Alexander; Faulstich, Konrad; Aivazachvili, Vissarion; Granger, Claire; Chenchik, Alex pp. 31-54 4. Preparing Fluorescent Probes for Microarray Studies Xiang, Charlie C.; Brownstein, Michael J. pp. 55-60 5. Escherichia coli Spotted Double-Strand DNA Microarrays: RNA Extraction, Labeling, Hybridization, Quality Control, and Data Management Khodursky, Arkady B.; Bernstein, Jonathan A.; Peter, Brian J.; Rhodius, Virgil; Wendisch, Volker F.; Zimmer, Daniel P. pp. 61-78 6. Isolation of Polysomal RNA for Microarray Analysis Arava, Yoav pp. 79-88 7. Parallel Analysis of Gene Copy Number and Expression Using cDNA Microarrays Pollack, Jonathan R. pp. 89-98 8. Genome-wide Mapping of Protein-DNA Interactions by Chromatin Immunoprecipitation and DNA Microarray Hybridization Lieb, Jason D. pp. 99-110 9. Statistical Issues in cDNA Microarray Data Analysis Smyth, Gordon K.; Yang, Yee Hwa; Speed, Terry pp. 111-136 10. Experimental Design to Make the Most of Microarray Studies Kerr, M. Kathleen pp. 137-148 11. Statistical Methods for Identifying Differentially Expressed Genes in DNA Microarrays Storey, John D.; Tibshirani, Robert pp. 149-158 12. Detecting Stable Clusters Using Principal Component Analysis Ben-Hur, Asa; Guyon, Isabelle pp. 159-182 13. Clustering in Life Sciences Zhao, Ying; Karypis, George pp. 183-218 14. A Primer on the Visualization of Microarray Data Fawcett, Paul pp. 219-234 15. Microarray Databases: Storage and Retrieval of Microarray Data Sherlock, Gavin; Ball, Catherine A. pp. 235-248
- 8. Fabrication of cDNA Microarrays 1 1 From: Methods in Molecular Biology: vol. 224: Functional Genomics: Methods and Protocols Edited by: M. J. Brownstein and A. Khodursky © Humana Press Inc., Totowa, NJ 1 Fabrication of cDNA Microarrays Charlie C. Xiang and Michael J. Brownstein 1. Introduction DNA microarray technology has been used successfully to detect the expression of many thousands of genes, to detect DNA polymorphisms, and to map genomic DNA clones (1–4). It permits quantitative analysis of RNAs transcribed from both known and unknown genes and allows one to compare gene expression patterns in normal and pathological cells and tissues (5,6). DNA microarrays are created using a robot to spot cDNA or oligonucleotide samples on a solid substrate, usually a glass microscope slide, at high densities. The sizes of spots printed in different laboratories range from 75 to 150 µm in diameter. The spacing between spots on an array is usually 100–200 µm. Microarrays with as many as 50,000 spots can be easily fabricated on standard 25 mm × 75 mm glass microscope slides. Two types of spotted DNA microarrays are in common use: cDNA and synthetic oligonucleotide arrays (7,8). The surface onto which the DNA is spotted is critically important. The ideal surface immobilizes the target DNAs, and is compatible with stringent probe hybridization and wash conditions (9). Glass has many advantages as such a support. DNA can be covalently attached to treated glass surfaces, and glass is durable enough to tolerate exposure to elevated temperatures and high-ionic-strength solutions. In addition, it is nonporous, so hybridization volumes can be kept to a minimum, enhancing the kinetics of annealing probes to targets. Finally, glass allows probes labeled with two or more fluors to be used, unlike nylon membranes, which are typically probed with one radiolabeled probe at a time.
- 9. 2 Xiang and Brownstein 2. Materials 1. Multiscreen filtration plates (Millipore, Bedford, MA). 2. Qiagen QIAprep 96 Turbo Miniprep kit (Qiagen, Valencia, CA). 3. dATP, dGTP, dCTP, and dTTP (Amersham Pharmacia, Piscataway, NJ). 4. M13F and M13R primers (Operon, Alameda, CA). 5. Taq DNA polymerase and buffer (Invitrogen, Carlsbad, CA). 6. PCR CyclePlate (Robbins, Sunnyvale, CA). 7. CycleSeal polymerase chain reaction (PCR) plate sealer (Robbins). 8. Gold Seal microscope slides (Becton Dickinson, Franklin, NJ). 9. 384-well plates (Genetix, Boston, MA). 10. Succinic anhydride (Sigma, St. Louis, MO) in 325 mL of 1-methy-2-pyrrolidinone (Sigma). 3. Methods 3.1. Selection and Preparation of cDNA Clones 3.1.1. Selection of Clones Microarrays are usually made with DNA fragments that have been amplified by PCR from plasmid samples or directly from chromosomal DNA. The sizes of the PCR products on our arrays range from 0.5 to 2 kb. They attach well to the glass surface. The amount of DNA deposited per spot depends on the pins chosen for printing, but elements with 250 pg to 1 ng of DNA (up to 9 × 108 molecules) give ample signals. Many of the cDNA clones that have been arrayed by laboratories in the public domain have come from the Integrated Molecular Analysis of Genomes and Expression (IMAGE) Consortium set. Five million human IMAGE clones have been collected and are available from Invitrogen/Research Genetics (www.resgen.com/products/IMAGEClones.php3). Sequence-verified cDNA clones from humans, mice, and rats are also available from Invitrogen/Research Genetics. cDNA clones can also be obtained from other sources. The 15,000 National Institute of Aging (NIA) mouse cDNA set has been distributed to many aca- demic centers (http://lgsun.grc.nia.nih.gov/cDNA/15k/hsc.html). Other mouse cDNA collections include the Brain Molecular Anatomy Project (BMAP) (http://brainest.eng.uiowa.edu), and RIKEN (http://genome.rtc.riken.go.jp) clone sets. In preparing our arrays, we have used the NIA and BMAP collec- tions and are in the process of sequencing the 5′ ends of the 41,000 clones in the combined set in collaboration with scientists at the Korea Research Institute of Bioscience and Biotechnology. Note that most cDNA collections suffer from some gridding errors and well-to-well cross contamination.
- 10. Fabrication of cDNA Microarrays 3 3.1.2. Preparation of Clones Preparing DNA for spotting involves making plasmid minipreps, amplifying their inserts, and cleaning up the PCR products. Most IMAGE clones are in standard cloning vectors, and the inserts can be amplified with modified M13 primers. The sequences of the forward (M13F) and reverse (M13R) primers used are 5′-GTTGTAAAACGACGGCCAGTG-3′ and 5′-CACACAGGAAA CAGCTATG-3′, respectively. A variety of methods are available for purifying cDNA samples. We use QIAprep 96 Turbo Miniprep kits and a Qiagen BioRobot 8000 (Qiagen) for plasmid isolations but cheaper, semiautomated techniques can be used as well. We PCR DNAs with a Tetrad MultiCycler (MJ Research, Incline Village, NV) and purify the products with Multiscreen filtration plates (Millipore). 3.1.3. Purification of Plasmid 1. Culture the bacterial clones overnight in 1.3 mL of Luria–Bertani (LB) medium containing 100 µg/mL of carbenicillin at 37°C, shaking them at 300 rpm in 96-well flat-bottomed blocks. 2. Harvest the bacteria by centrifuging the blocks for 5 min at 1500g in an Eppendorf centrifuge 5810R (Eppendorf, Westbury, NY). Remove the LB by inverting the block. The cell pellets can be stored at –20°C. 3. Prepare cDNA using the BioRobot 8000, or follow the Qiagen QIAprep 96 Turbo Miniprep kit protocol for manual extraction. 4. Elute the DNA with 100 µL of Buffer EB (10 mM Tris-HCl, pH 8.5) included in the QIAprep 96 Turbo Miniprep kit. The plasmid DNA yield should be 5–10 µg per prep. 3.1.4. PCR Amplification 1. Dilute the plasmid solution 1⬊10 with 1X TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). 2. For each 96-well plate to be amplified, prepare a PCR reaction mixture containing the following ingredients: 1000 µL of 10X PCR buffer (Invitrogen), 20 µL each of dATP, dGTP, dCTP, and dTTP (100 mM each; Amersham Pharmacia), 5 µL each of M13F and M13R (1 mM each; Operon), 100 µL of Taq DNA polymerase (5 U/µL; Invitrogen), and 8800 µL of ddH2O. 3. Add 100 µL of PCR reaction mix to each well of a PCR CyclePlate (Robbins) plus 5 µL of diluted plasmid template. Seal the wells with CycleSeal PCR plate sealer (Robbins). (Prepare two plates for amplification from each original source plate to give a final volume of 200 µL of each product.) 4. Use the following PCR conditions: 96°C for 2 min; 30 cycles at 94°C for 30 s, 55°C for 30 s, 72°C for 1 min 30 s; 72°C for 5 min; and cool to ambient temperature.
- 11. 4 Xiang and Brownstein 5. Analyze 2 µL of each product on 2% agarose gels. We use an Owl Millipede A6 gel system (Portsmouth, NH) with eight 50-tooth combs. This allows us to run 384 samples per gel. 3.1.5. Cleanup of PCR Product 1. Transfer the PCR products from the two duplicate PCR CyclePates to one Millipore Multiscreen PCR plate using the Qiagen BioRobot 8000. 2. Place the Multiscreen plate on a vacuum manifold. Apply the vacuum to dry the plate. 3. Add 100 µL of ddH2O to each well. 4. Shake the plate for 30 min at 300 rpm. 5. Transfer the purified PCR products to a 96-well plate. 6. Store the PCR products in a –20°C freezer. 3.2. Creating cDNA Microarrays (see Note 1) Robots are routinely used to apply DNA samples to glass microscope slides. The slides are treated with poly-L-lysine or other chemical coatings. Some investigators irradiate the printed arrays with UV light. Slides coated with poly-L-lysine have a positively charged surface, however, and the negatively charged DNA molecules bind quite tightly without crosslinking. Finally, the hydrophobic character of the glass surface minimizes spreading of the printed spots. Poly-L-lysine-coated slides are inexpensive to make, and we have found that they work quite well. About 1 nL of PCR product is spotted per element. Many printers are commercially available. Alternatively, one can be built in-house (for detailed instructions, visit http://cmgm.stanford.edu/pbrown/mguide/index.html). After the arrays are printed, residual amines are blocked with succinic anhydride (see http://cmgm.stanford.edu/pbrown/mguide/index.html). 3.2.1. Coating Slides with Poly-L-lysine 1. Prepare cleaning solution by dissolving 100 g of NaOH in 400 mL of ddH2O. Add 600 mL of absolute ethanol and stir until the solution clears. 2. Place Gold Seal microscope slides (Becton Dickinson) into 30 stainless-steel slide racks (Wheaton, Millville, NJ). Place the racks in a glass tank with 500 mL of cleaning solution. Work with four racks (120 slides in total) at a time. 3. Shake at 60 rpm for 2 h. 4. Wash with ddH2O four times, 3 min for each wash. 5. Make a poly-L-lysine solution by mixing 80 mL of 0.1% (w/v) poly-L-lysine with 80 mL of phosphate-buffered saline and 640 mL of ddH2O. 6. Transfer two racks into one plastic tray with 400 mL of coating solution. 7. Shake at 60 rpm for 1 h.
- 12. Fabrication of cDNA Microarrays 5 8. Rinse the slides three times with ddH2O. 9. Dry the slides by placing them in racks (Shandon Lipshaw, Pittsburgh, PA) and spinning them at 130g for 5 min in a Sorvall Super T21 centrifuge with an ST-H750 swinging bucket rotor. Place one slide rack in each bucket. 10. Store the slides in plastic storage boxes and age them for 2 wk before printing DNA on them. 3.2.2. Spotting DNA on Coated Slides We use the following parameters to print 11,136 element arrays with an OmniGrid robot having a Server Arm (GeneMachines, San Carlos, CA): 4 × 4 SMP3 pins (TeleChem, Sunnyvale, CA), 160 × 160 µM spacing, 27 × 26 spots in each subarray, single dot per sample. We use the following printing parameters: velocity of 13.75 cm/s, acceleration of 20 cm/s2, decelera- tion of 20 cm/s2. We print two identical arrays on each slide. 1. Adjust the relative humidity of the arrayer chamber to 45–55% and the tempera- ture to 22°C. 2. Dilute the purified PCR products 1⬊1 with dimethylsulfoxide (DMSO) (Sigma) (see Note 2). Transfer 10-µL aliquots of the samples to Genetix 384-well plates (Genetix). 3. Load the plates into the cassette of the Server Arm. Three such cassettes hold 36 plates. Reload the cassettes in midrun if more than 36 plates of samples are to be printed. It takes about 24 h to print 100 slides with 2 × 11,136 elements on them. 4. Label the slides. Examine the first slide in the series under a microscope. Mark the four corners of the array (or the separate arrays if there are more than one on the slide) with a scribe. Use this indexed slide to draw a template on a second microscope slide showing where the cover slip should be placed during the hybridization step. Remove the remaining slides from the arrayer and store them in a plastic box. 3.2.3. Postprocessing We often postprocess our arrays after storing them for several days. This may not be necessary as others have argued, but it is sometimes convenient. Many workers recommend UV crosslinking the DNA to the slide surface by exposing the arrays to 450 mJ of UV irradiation in a Stratalinker (Stratagene, La Jolla, CA). As noted, this step is optional, and we have not found it to be critical. 1. Insert 30 slides into a stainless steel rack and place each rack in a small glass tank. 2. In a chemical fume hood, dissolve 6 g of succinic anhydride (Sigma) in 325 mL of 1-methy-2-pyrrolidinone (Sigma) in a glass beaker by stirring.
- 13. 6 Xiang and Brownstein 3. Add 25 mL of 1 M sodium borate buffer (pH 8.0) to the beaker as soon as the succinic anhydride is dissolved. 4. Rapidly pour the solution into the glass tank. 5. Place the glass tank on a platform shaker and shake at 60 rpm for 20 min in the hood. While the slides are incubating on the shaker, prepare a boiling water bath. 6. Transfer the slides to a container with 0.1% sodium dodecyl sulfate solution. Shake at 60 rpm for 3 min. 7. Wash the slides with ddH2O for 2 min. Repeat the wash two more times. 8. Place the slides in the boiling water bath. Turn off the heat immediately after submerging the slides in the water. Denature the DNA for 2 min in the water bath. 9. Transfer the slides to a container with 100% ethanol and incubate for 4 min. 10. Dry the slides in a centrifuge at 130g for 5 min (see Subheading 3.2.1., step 9) and store them in a clean plastic box. The slides are now ready to be probed (see Note 3). 4. Notes 1. The methods for printing slides described in this chapter are somewhat tedious, but they are robust and inexpensive. 2. We recommend dissolving the DNAs to be printed in 50% DMSO instead of aqueous buffers because this is a simple solution to the problem of sample evaporation during long printing runs (10). 3. The probe-labeling technique that we describe in Chapter 4 works well with slides prepared according to the protocols we have given. References 1. Schena, M., Shalon, D., Davis, R. W., and Brown, P. O. (1995) Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270, 467–470. 2. Schena, M., Shalon, D., Heller, R., Chai, A., Brown, P. O., and Davis, R. W. (1996) Parallel human genome analysis: microarray-based expression monitoring of 1000 genes. Proc. Natl. Acad. Sci. USA 93, 10,614–10,619. 3. DeRisi, J.,Vishwanath, R. L., and Brown, P. O. (1997) Exploring the metabolic and genetic control of gene expression on a genomic scale. Science 278, 680–686. 4. Sapolsky, R. J. and Lipshutz, R. J. (1996) Mapping genomic library clones using oligonucleotide arrays. Genomics 33, 445–456. 5. DeRisi, J., Penland, L., Brown, P. O., Bittner, M. L., Meltzer, P. S., Ray, M., Chen, Y., Su, Y. A., and Trent, J. M. (1996) Use of a cDNA microarray to analyse gene expression patterns in human cancer. Nat. Genet. 14, 457–460. 6. Heller, R. A., Schena, M., Chai, A., Shalon, D., Bedilion, T., Gilmore, J., Wool- ley, D. E., and Davis, R. W. (1997) Discovery and analysis of inflammatory disease-related genes using cDNA microarrary. Proc. Natl. Acad. Sci. USA 94, 2150–2155.
- 14. Fabrication of cDNA Microarrays 7 7. Shalon, D., Smith, S. J., and Brown, P. O. (1996) A DNA microarray system for analyzing complex DNA samples using two-color fluorescent probe hybridization. Genome Res. 6, 639–645. 8. Lipshutz, R. J., Fodor, S. P. A., Gingeras, T. R., and Lockhart, D. J. (1999). High density synthetic oligonucleotide arrays. Nat. Genet. 21(Suppl.), 20–24. 9. Cheung, V. G., Morley, M., Aguilar, F., Massimi, A., Kucherlapati, R., and Childs, G. (1999) Making and reading microarrays. Nat. Genet. 21(Suppl.), 15–19. 10. Hegde, P., Qi, R., Abernathy, K., Gay, C., Dharap, S., Gaspard, R., Hughes, J. E., Snesrud, E., Lee, N., and Quackenbush, J. (2000) A concise guide to cDNA microarray analysis. Biotechniques 29, 548–556.
- 15. 8 Xiang and Brownstein
- 16. Nylon cDNA Expression Arrays 9 9 From: Methods in Molecular Biology: vol. 224: Functional Genomics: Methods and Protocols Edited by: M. J. Brownstein and A. Khodursky © Humana Press Inc., Totowa, NJ 2 Nylon cDNA Expression Arrays George Jokhadze, Stephen Chen, Claire Granger, and Alex Chenchik 1. Introduction Nucleic acid arrays provide a powerful methodology for studying biological systems on a genomic scale. BD Atlas™ Arrays, developed by BD Biosciences Clontech, are expression profiling products specifically designed to be acces- sible to all laboratories performing isotopic blot hybridization experiments. We have developed two types of readily accessible BD Atlas Arrays: nylon macroarrays, well suited for high-sensitivity expression profiling using a limited gene set, and broad-coverage plastic microarrays, for a more extensive analysis of a comprehensive set of genes. In this chapter, we describe protocols for printing and performing gene expression analysis using nylon membrane–based arrays. For a more in-depth description and protocols related to plastic film–based arrays, please refer to Chapter 3. Nylon membrane–based arrays offer several advantages for researchers. Compared with glass arrays, nylon arrays are usually less expensive to produce and require less complicated equipment. Nylon arrays are generally considered more user friendly, since analysis involves only familiar hybridiza- tion techniques. Detection of results is also straightforward—probes are radioactively labeled, so one can simply use a standard phosphorimager. 1.1. Sensitivity of Nylon Arrays Nylon membranes are typically used to print low- (10–1000) to medium- (1000–4000) density cDNA arrays. Unlike high-density arrays, which are usually printed on glass or plastic supports, probes for nylon arrays can be labeled with 32P, resulting in a much higher (>fourfold) level of sensitivity
- 17. 10 Jokhadze et al. (Fig. 1). This means that the presence of even low-abundance transcripts can be detected. Nylon arrays are printed with fragments of cDNA clones (200–600 bp) representing individual genes. Each cDNA fragment is amplified from the original clone using gene-specific or universal primers, denatured, and printed onto the membranes. cDNA fragments have a significantly higher hybridization efficiency than oligos yet generally do not allow discrimination between highly homologous genes, such as multigene family members. For this reason, cDNA fragments are ideal for nylon arrays that represent a limited number of genes. In an array experiment, the cDNA fragments on the array are designated as the “targets.” The “probe” used to screen the array is a radioactively labeled pool of cDNAs, reverse transcribed from total or polyA+ RNA extracted from a particular tissue or cell type. Duplicate arrays are screened with cDNA probes prepared from two or more tissues, cell lines, or differentially treated samples. The single most important factor determining the success or failure of array experiments is the quality of the RNA used to make the probes. Poor- quality RNA preparation leads to high background on the membrane and/or a misleading hybridization pattern. The present protocol allows purification of total RNA and labeling of probes for array hybridizations in one straightforward procedure—no separate polyA+ RNA purification step is needed.An acceptable Fig. 1. Nylon array hybridized with a 32P-labeled probe.
- 18. Nylon cDNA Expression Arrays 11 amount (10 µg) of high-quality total RNA can be isolated from as little as 10 mg of tissue or 105 cells. With nylon membrane arrays, there is a choice of using 32P or 33P in the labeling reaction. The more appropriate method depends on the printing density of the array (see Subheading 3.1.4.) and the nature of the experiment. For general purposes, we recommend using 32P because this isotope provides greater sensitivity. High sensitivity will be especially important if one is interested in any low-abundance transcripts. On the other hand, 33P offers the advantage of higher-resolution signal, meaning that the signal produced by a spot on the array will be more closely confined to the spot’s center, preventing signal “bleed” to neighboring spots. High signal bleed can complicate the interpretation of results for nearby genes. The 33P method is particularly useful if highly abundant transcripts are of interest or one plans to quantitatively analyze the results by phosphorimaging. However, 33P detection is generally only one-fourth as sensitive as 32P detection (1). When labeling array probes, choose the method that best suits your needs. 2. Materials Unless otherwise noted, all catalog numbers provided are for BD Biosciences Clontech products. 2.1. Nylon Membrane Array Printing 2.1.1. Nylon Membrane Printing Reagents 1. Nytran Plus Membrane, cut into 82 × 120 mm rectangles (Schleicher & Schuell). 2. BD TITANIUM™ Taq PCR Kit (cat. no. K1915-1). 3. Gene-specific or universal primers for amplifying cDNA fragments (see Subheading 3.1.). 4. Sequence-verified cDNA templates (vectors carrying clones with sequence- verified cDNA insert). 5. Milli-Q-filtered H2O. 6. Printing dye (30% Ficoll, 1% thymol blue). 7. 3 M NaOAc, pH 4.0. 8. Membrane neutralization solution (0.5 M Tris, pH 7.6). 2.1.2. Nylon Membrane Array Printing Equipment 1. Polymerase chain reaction (PCR) reaction tubes (0.5 mL). (We recommend Perkin-Elmer GeneAmp 0.5-mL reaction tubes (cat. no. N801-0737 or N801-0180). 2. PCR machine/thermal cycler. We use a hot-lid thermal cycler.
- 19. 12 Jokhadze et al. 3. 384-well V-bottomed polystyrene plates (USA Scientific), for use as a source plate during printing. 4. SpeedVac. 5. Arrayer robot. We use a BioGrid Robot (BioRobotics). 6. UV Stratalinker crosslinker (Stratagene). 7. Pin tool (0.7 mm diameter, 384 pin). 8. Sarstedt Multiple Well Plate 96-Well (lids only), used to hold nylon membranes for printing. 9. Adhesive sealing film (THR100 Midwest Scientific). 10. NucleoSpin Multi-8 PCR Kit (cat. no. K3059-1) or NucleoSpin Multi-96 PCR Kit (cat. no. K3065-1). 2.2. Reagents for RNA Isolation and Probe Synthesis 2.2.1. Reagents Provided with BD Atlas Pure Total RNA Labeling System The BD Atlas™ Pure Total RNA Labeling System (cat. no. K1038-1) is available exclusively from BD Biosciences Clontech. Do not use the protocol supplied with the BD Atlas Pure Kit. The procedures for RNA isolation and cDNA synthesis in the following protocol differ significantly from the procedures found in the BD Atlas Pure User Manual. 1. Denaturing solution. 2. Saturation buffer for phenol. 3. RNase-free H2O. 4. 2 M NaOAc (pH 4.5). 5. 10X termination mix. 6. Streptavidin magnetic beads. 7. 1X binding buffer. 8. 2X binding buffer. 9. 1X reaction buffer. 10. 1X wash buffer 11. DNase I (1 U/µL). 12. DNase I buffer. 13. Biotinylated oligo(dT). 14. Moloney murine leukemia virus reverse transcriptase (MMLV RT). 2.2.2. Additional Reagents/Special Equipment 1. Saturated phenol (store at 4°C). For 160 mL: 100 g of phenol (Sigma cat. no. P1037 or Boehringer Mannheim cat. no. 100728). In a fume hood, heat a jar of phenol in a 70°C water bath for 30 min or until the phenol is completely melted. Add 95 mL of phenol directly to the saturation buffer (from the BD AtlasPure Kit), and mix well. Hydroxyquinoline may be added if desired. Aliquot and freeze at –20°C for long-term storage. This preparation of saturated phenol will only have one phase.
- 20. Nylon cDNA Expression Arrays 13 2. Tissue homogenizer (e.g., Polytron or equivalent). For <200 mg of tissue, use a 6-mm probe. For >200 mg of tissue, use a 10-mm probe. 3. [α-32P]dATP (10 µCi/µL; 3000 Ci/mmol) (cat. no. PB10204; Amersham) or [α-33P]dATP (10 µCi/µL; >2500 Ci/mmol) (cat. no. BF1001; Amersham). Do not use Amersham’s Redivue or any other dye-containing isotope. 4. Deionized H2O (Milli-Q filtered or equivalent; do not use diethylpyrocarbonate- treated H2O). 5. Magnetic particle separator (cat. no. Z5331; Promega, Madison, WI). It is important that you use a separator designed for 0.5-mL tubes. 6. Polypropylene centrifuge tubes: 1.5-mL (cat. no. 72-690-051; Sarstedt), 2-mL (cat. no. 16-8105-75; PGC), 15-mL (tubes cat. no. 05-562-10D, caps cat. no. 05-562-11E; Fisher), and 50-mL (tubes with caps cat. no. 05-529-1D; Fisher). Fifteen- and 50-mL tubes should be sterilized with 1% sodium dodecyl sulfate (SDS) and ethanol before use. 7. 10X dNTP mix (for dATP label; 5 mM each of dCTP, dGTP, dTTP). 8. 10X Random primer mix (N-15) or gene-specific primer mix (see Subhead- ing 3.4.3.). 9. BD PowerScript™ Reverse Transcriptase and 5X BD PowerScript™ Reaction Buffer (available exclusively from BD Biosciences Clontech; cat. no. 8460-1). 10. Dithiothreitol (DTT) (100 mM). 11. NucleoSpin® Extraction Kit: NucleoSpin extraction spin columns, 2-mL collec- tion tubes, buffer NT2, buffer NT3 (add 95% ethanol before use as specified on the label), buffer NE. 2.3. Reagents for Hybridization, Washing, and Stripping of Nylon Arrays 1. BD ExpressHyb™ hybridization solution (cat. no. 8015-1). 2. Sheared salmon testes DNA (10 mg/mL) (cat. no. D7656; Sigma). 3. Optional: 10X Denaturing solution (1 M NaOH, 10 mM EDTA) (see Subhead- ing 3.5.). 4. Optional: 2X Neutralizing solution (1 M NaH2PO4 [pH 7.0]): 27.6 g of NaH2PO4•H2O). Add 190 mL of H2O, adjust the pH to 7.0 with 10 N NaOH if necessary, and add H2O to 200 mL. Store at room temperature (see Subhead- ing 3.5.). 5. Cot-1 DNA (1 mg/mL). 6. 20X saline sodium citrate (SSC), 175.3 g of NaCl, 88.2 g of Na3citrate•2H2O. Add 900 mL of H2O, adjust the pH to 7.0 with 1 M HCl if necessary, and add H2O to 1 L. Store at room temperature. 7. 20% SDS: 200 g of SDS. Add H2O to 1 L. Heat to 65°C to dissolve. Store at room temperature. 8. Wash solution 1: 2X SSC, 1% SDS. Store at room temperature. 9. Wash solution 2: 0.1X SSC, 0.5% SDS. Store at room temperature.
- 21. 14 Jokhadze et al. 3. Methods 3.1. Printing of Nylon Membrane Arrays cDNA fragments to be used for printing can be amplified by using either gene-specific primers or a pair of “universal” primers (i.e., T3, T7, M13F, or M13R) complementary to sites in the cloning vector flanking the cDNA clone. One advantage of using gene-specific primers is that a specific region of the cDNA clone to be amplified can be chosen. For example, the amplification of cDNAs used to print BD Atlas Arrays is specially designed to minimize nonspecific hybridization. All cDNA fragments are 200–600 bp long and are amplified from a region of the mRNA that lacks the poly A tail, repetitive elements, or other highly homologous sequences. Another advantage of using gene-specific primers is that the antisense primers used in array preparation can be pooled and subsequently used as a gene-specific primer mix to synthesize cDNA probes from experimental samples. The use of gene-specific probes provides higher sensitivity and lower background than random primers (see Subheading 3.4.3. for details). 3.1.1. Preparative PCR for cDNA Fragments 1. Prepare a 100-µL PCR reaction in a 0.5-mL PCR tube for each cDNA to be represented on the array. Calculate the amount of each component required for the PCR reaction by referring to Table 1. Universal primers, appropriate for your cloning vector, may be used in place of gene-specific primers. Adjust the volumes accordingly. 2. Commence thermal cycling using the following parameters: 30–35 cycles of 94°C for 30 s and 68°C for 90 s, 68°C for 5 min, and 15°C soak. These conditions were developed for use with a hot-lid thermal cycler; the optimal parameters may vary with different thermal cyclers. (Note that these parameters were optimized for amplification of fragments approx 200–600 bp long.) 3. Run 5 µL of each pooled PCR product (plus loading dye) on a 2% TAE agarose gel, alongside a molecular weight marker, to screen the PCR products. 4. Check each PCR product size by comparison with the molecular weight markers. If the size of the PCR product is correct, add EDTA (final concentration of 0.1 M EDTA, pH 8.0) to the pooled PCR products to stop the reaction. 3.1.2. Purification of cDNA Fragments To purify amplified cDNA fragments, we recommend that you use either the NucleoSpin Multi-8 PCR Kit (cat. no. K3059-1) or NucleoSpin Multi-96 PCR Kit (cat. no. K3065-1) and follow the enclosed protocol. NucleoSpin PCR kits are designed to purify PCR products from reaction mixtures with speed and efficiency. Primers, nucleotides, salts, and polymerases are effectively removed using these kits; up to 96 samples can be processed simultaneously in less than
- 22. Nylon cDNA Expression Arrays 15 60 min. Up to 15 µg of high-quality DNA can be isolated per preparation. Recovery rates of 75–90% can be achieved for fragments from 100 bp to 10 kb. 3.1.3. Standardization of cDNAs 1. In a 1.5-mL microcentrifuge tube, dilute 5 µL of the purified cDNA fragment stock in 995 µL of H2O (a 1⬊200 dilution) and read the optical density of the dilution at 260 nm. Calculate the cDNA concentration in cDNA stock. Each PCR reaction should yield a total of 2 to 3 µg of DNA. 2. If the concentration of cDNA in the stock solution is >500 ng/µL, go to step 5; if <500 ng/µL, continue with the next step. 3. Concentrate the cDNA stock solution by evaporation in a SpeedVac. Repeat steps 1 and 2. 4. Adjust the concentration to 500 ng/µL by adding Milli-Q-H2O: VH 2 O = (Ci × Vi/Cf) – Vi, in which Ci and Vi are the initial concentration and volume of the main solution (before adding H2O), respectively; and Cf is the final, desired concentration. 5. Store the normalized cDNA at –20°C. 3.1.4. Printing of cDNA Arrays on Nylon Membranes An 80 mm × 120 mm rectangle of nylon membrane can be printed with as many as 3000 cDNA fragments (using a 384-pin tool with 0.7-mm-diameter pins) without encountering significant difficulties with image analysis due to signal bleed. If 32P-labeled probes are used, the maximum printing density on a membrane of the same size should be no more than 1500, to avoid loss of signal resolution. Depending on your experimental needs and organism, you may wish to include negative controls, such as genomic DNA, phage lambda DNA, or yeast Table 1 cDNA Fragment PCR Set-Up Per 100-µL reaction PCR master mix Final concentration (µL) 10X BD TITANIUM Taq 1X 10 PCR buffer 10 µM dNTP mix 200 µM 2 Specific or universal 0.4 µM each 2 primer mix, 20 µM each Template (0.5–1 ng/µL) 0.025–0.05 ng/µL 5 50X BD TITANIUM Taq Mix 1X 2 Milli-Q H2O Bring volume up 79
- 23. 16 Jokhadze et al. DNA. Some researchers also choose to include cDNA fragments representing certain housekeeping genes, known to be highly expressed in their experimental samples, to serve as positive controls. 1. Prepare the individual cDNA printing mixes. The final cDNA concentration for printing should be approx 100 ng/µL. The final NaOH concentration for printing should be 0.15 N. The final printing dye concentration for printing should be 1X. The volume of solution deposited by a single, 0.7-mm-diameter pin is 90 nL, which is equivalent to 10 ng of cDNA printed per spot. For example, to prepare 25 µL of ready-to-print cDNA solution with a ~110 ng/µL final concentration, combine: 5.5 µL of cDNA (500 ng/µL), 0.4 µL of 10 N NaOH, 2.5 µL of 10X dye, and 16.6 µL of Milli-Q H2O, for a total of 25.0 µL. This volume is sufficient for printing approx 200 arrays with single spots for each cDNA, or 100 arrays with duplicate spots. (Printing from volumes of <2 to 3 µL may result in irregular spot morphology.) 2. Aliquot 25 µL of each cDNA printing mix into individual wells of a 384-well plate. 3. Prepare the arrayer for printing following the manufacturer’s user manual. (We use a BioRobotics BioGrid.) 4. Place each nylon membrane onto a lid from a Sarstedt 96-well plate. This will hold the membrane securely during printing. Place the Nytran Plus membranes and lids into the filter tray (the Biogrid tray holds 24 membranes at a time). 5. Begin the printing process according to the manufacturer’s instructions. 6. Replace the water and ethanol in the arrayer’s trays after every second round of printing. 7. After the completion of printing, allow the membranes to dry for 45 min at room temperature. 8. Using forceps, pick up the dried, printed membranes, grasping each membrane only by the edge, and drop into a tray containing membrane-neutralizing solution. Gently agitate the membrane arrays for approx 1 min. Change the solution after every 48 membranes. 9. Crosslink the membranes using an energy of 120 mJ/cm2 (1200 × 100 µJ/cm2) in a UV Stratalinker Crosslinker. When complete, remove the membranes from the Stratalinker and lay flat to dry for at least 4 h. Dried arrays should be stored at –20°C, sealed individually in plastic bags. 3.2. RNA Isolation 3.2.1. RNA Isolation from Tissues Conical 50-mL tubes can break under forces >10,000g. We recommend using sterile 15- and 50-mL round-bottomed, polypropylene centrifuge tubes at all times. 1. Harvest the tissue; use immediately or flash freeze in liquid nitrogen and store at –70°C. Important: When working with frozen tissue, be sure to keep the
- 24. Nylon cDNA Expression Arrays 17 tissue frozen until you add the denaturing solution. Even partial thawing can result in RNA degradation. Perform all necessary manipulations on dry ice or liquid nitrogen. 2. Cut or crush the tissue into small pieces (<1 cm3). When working with frozen tissue, prechill a mortar and pestle with liquid nitrogen, fill the mortar with liquid nitrogen, and break frozen tissue into smaller pieces. 3. Weigh out the tissue in a prechilled, sterile tube. See Table 2 for the appropriate tube size. 4. Add the appropriate volume (see Table 2) of denaturing solution. Always add at least 1 mL/100 mg of tissue. 5. Grind the sample at 0–4°C using a tissue homogenizer (e.g., Polytron or equiva- lent) at the maximum setting for 1 to 2 min or until completely homogenized. 6. Incubate on ice for 5–10 min. 7. Vortex the sample thoroughly. Centrifuge the homogenate at 15,000g for 5 min at 4°C to remove cellular debris. 8. Transfer the entire supernatant to new centrifuge tube(s). Avoid pipeting the insoluble upper layer, if present. 9. Add the appropriate volume (see Table 2) of saturated phenol. 10. Cap the tubes securely and vortex for 1 min. Incubate on ice for 5 min. 11. Add the appropriate volume (see Table 2) of chloroform. 12. Shake the sample and vortex vigorously for 1 to 2 min. Incubate on ice for 5 min. 13. Centrifuge the homogenate at 15,000g for 10 min at 4°C. 14. Transfer the upper aqueous phase containing the RNA to a new tube. Take care not to pipet any material from the white interface or lower organic phase. 15. Perform a second round of phenol:chloroform extraction, using the amounts shown in Table 2 for “2nd round” (see Note 1). Repeat steps 9–14. Table 2 Reagents for RNA Isolation from Tissues Weight of tissue 10–100 mg 100–300 mg 300–600 mg 0.6–1.0 g Recommended tube size (mL) 2 × 2 15a.1 50a.1 50a11 Denaturing solution (mL) 1.0 13.0a 16.0a 10.0a Saturated phenol (mL) 2.0 16.0a 12.0a 20.0a Chloroform (mL) 0.6 11.8a 13.6a 16.0a Saturated phenol (2nd round) (mL) 1.6 14.8a 19.6a 16.0a Chloroform (2nd round) (mL) 0.6 11.8a 13.6a 16.0a Isopropanol (mL) 2.0 16.0a 12.0a 20.0a 80% EtOH wash (mL) 1.0 13.0a 16.0a 10.0a aConical tubes can break under forces greater than 10,000g. Ensure that round-bottomed tubes are used.
- 25. 18 Jokhadze et al. 16. Transfer the upper phase to a new tube. Avoid touching the interface. 17. Add the appropriate volume (see Table 2) of isopropanol. Add slowly, mixing occasionally as you add it. 18. Mix the solution well and incubate on ice for 10 min. 19. Centrifuge the samples at 15,000g for 15 min at 4°C. 20. Quickly remove the supernatant without disturbing the RNA pellet. 21. Add the appropriate volume (see Table 2) of 80% ethanol. 22. Centrifuge at 15,000g for 5 min at 4°C. Quickly and carefully discard the supernatant. 23. Air-dry the pellet. 24. Resuspend the pellet in enough RNase-free H2O to ensure an RNA concentration of 1 to 2 µg/µL. Refer to Table 4 for approximate yields. 25. Allow the pellet to soak, then resuspend thoroughly by tapping the tube and pipeting. 26. Set aside a 2-µL aliquot to compare with your RNA sample following DNase treatment. Store the RNA samples at –70°C until ready to proceed with DNase treatment. 3.2.2. RNA Isolation from Cultured Cells 1. Transfer the cultured cells to a sterile tube. See Table 3 for the appropriate tube size. 2. Centrifuge at 500g for 5 min at 4°C. Discard the supernatant. 3. Use the cells immediately, or flash freeze in liquid nitrogen and store at –70°C. When working with frozen cells, be sure to keep the cells frozen until you add Table 3 Reagents for RNA Isolation from Cultured Cells Cell number 106–107 1–3 × 107 3–6 × 107 6–10 × 107 Tube size (mL) 2 × 2 15a.. 50a1 50a1 Denaturing solution (mL) 1.0 13.0a 16.0a 10.0a Saturated phenol (mL) 2.0 16.0a 12.0a 20.0a Chloroform (mL) 0.6 11.8a 13.6a 16.0a Saturated phenol (2nd round) (mL) 1.6 14.8a 19.6a 16.0a Chloroform (2nd round) (mL) 0.6 11.8a 13.6a 16.0a Isopropanol (mL) 2.0 16.0a 12.0a 20.0a 80% EtOH wash (mL) 1.0 13.0a 16.0a 10.0a aConical tubes can break under forces greater than 10,000g. Ensure that round-bottomed tubes are used.
- 26. Other documents randomly have different content
- 27. Gavin pulled himself together with an effort and scrambled to his feet. "Curious," he admitted frankly. From the corner of his eyes, he saw Nadia sit up, holding her temples. Villanowski's eyes narrowed. "What did you see?" "Nothing," Gavin said in a disappointed voice. He could feel the perspiration break through his pores. Villanowski was no fool. "The mist caught us at the foot of the ladder." He paused. "Next time we go into the little death, I'd like to be in the control room with you." "You saw the control room?" "Oh, the door was open." Gavin summoned all his histrionic ability to sound convincing. "I saw you and started in. Then—then I fainted, I guess." Indecision was reflected on Villanowski's face. Finally he growled, "Get out, the pair of you. Don't come down here again except on your watch." Gavin followed the girl up the ladder, his palms slippery with sweat on the cold bars. He wasn't sure whether he'd fooled Villanowski or not. They entered the mess room, helped themselves to coffee. He realized the girl hadn't uttered a word since the little death. He saw she was regarding him with a half-frightened, half-perplexed frown. "How did the little death affect you?" he asked her. "I had a dream. At least, I think it was." She bit her lip. "What was it about?" "Something in the future." She laughed. "It's silly, isn't it, to be so frightened at a dream. Especially one so fantastic." "I don't know," Gavin replied dryly. "Mine didn't leave me so comfortable ... but you haven't told me what yours was."
- 28. Nadia rubbed her temples. "I dreamed we were all captured on Jupiter and sent to the Penal Colony." She laughed at herself. "It's so silly, because you—you"—again she gave a low laugh—"you were a T.I.S. agent!" Gavin felt his mouth go dry. He stared at her in consternation. He moistened his lips and started to ask for more particulars, when the chief astrogator entered the messroom. The Nova's astrogator nodded perfunctorily at them and went across to the solar chart. Very deliberately, he pulled out the pin marking the Nova's position, moved it across the map to within a week's voyage of Jupiter. Gavin couldn't believe his eyes. "That's impossible! We weren't unconscious but a few seconds during the little death...." "An hour," Nadia corrected. "The effects last an hour. So Villanowski claims." "An hour, then. What difference does it make? The Nova couldn't have gone that distance in an hour, nor in a thousand hours! Why man, that's faster than the speed of light!" The chief astrogator grunted. "Impossible or not, that's our position. If you don't think so, go on out on the observation deck and take a look." Gavin leaped to his feet and plunged through the door. When he came out on the quartzite enclosed deck, he flung his gaze aloft. The entire aspect of the heavens had changed. The three streaks denoting the pursuing space craft were absent. The sun had diminished to the size of a lemon. And dead ahead loomed the huge banded disc of Jupiter. It was true. In the space of a few moments the Nova had traversed the void between Venus and Jupiter. Even though the planets were in a superior conjunction, the feat was unthinkable. No wonder the big corporations were fighting like wildcats to get hold of the Nova's spacedrive!
- 29. Gavin dropped weakly in a deck chair, overawed by the possibilities. A new era of space travel was being inaugurated! VII Gavin Murdock was on watch below, when the Nova sliced into the upper strata of Jupiter's atmosphere. She dived in at a slant on the opposite side of the planet for Jovopolis and was quickly smothered from view by the thick translucent air. To the early astronomers, Jupiter had appeared enormous although it was only about a third denser than water. But the Huygen expedition in the first years of interplanetary travel had resolved the enigma. Jupiter consisted of a small solid core surrounded by an intensive and very dense atmosphere. The force of gravity at the surface was only between two and three times that on Terra. Nominally, Jupiter was a colony of the Terran empire. But every attempt at settlement had proved disastrous. Today Jovopolis was an outpost, consisting of rotting shacks, a trading post, and one modern structure which housed the Huygen Memorial Institute of Science. Even the interplanetary patrol had made no attempt to install a permanent base. The officers and men lived in their ship while they were assigned to the station. The Nova began to settle Jove-ward. Gavin never left the bridge televisor, pulling switches, relaying orders to the jetman and master mechanic as the clumsy monster performed the ticklish job of landing. Villanowski, who had been routed out of his bunk by the landing alarm, paced back and forth the length of the engine room, his eyes everywhere. He didn't interfere, though. Then the Nova struck with a bump which threw the chief engineer to his hands and knees.
- 30. He scrambled up, brushing off his shorts. "Nice landing." He patted Gavin's shoulder. "Nice landing." It was a nice landing. Gavin's bony freckled features relaxed. Broken ankles, bruises and sprains were only too frequent when setting a ship down without the benefit of spaceways. "How long will it take us to load?" "Five days," Villanowski replied. "Jovian time." The Jovian day, Gavin knew, was only nine hours and fifty-five minutes long. That meant the Nova would be on Jupiter forty-nine hours. Gavin made a hasty mental calculation. It cut the margin of success to the barest minimum time. He would have to act and act quickly. He started for the ladder, feeling the increased gravity tug at his flesh. "Not so fast, lad," said Villanowski. Gavin paused. The chief engineer's attitude had undergone such a remarkable change that Gavin's own suspicions had been aroused. It dated from the second meal following the little death. Nadia had regaled the officers with her dream—the one in which Gavin had been a T.I.S. agent and contrived the capture of them all on Jupiter. Villanowski's homely face had clouded. Then he had remarked with a wry expression that stranger things could happen. Oddly enough, however, he no longer evinced the slightest suspicion of Gavin. But an occasional joshing reference to Murdock, the T.I.S. agent, revealed that he hadn't forgotten Nadia's dream. Gavin said, "Yes sir," in a doubtful tone. He couldn't afford to arouse any doubts now. "The Captain wants to see you before you go ashore." "Yes, sir."
- 31. Wondering what Cabot wanted with him, he struggled up the ladder. Sweat began to pour from his skin. It was like climbing with the old man of the sea anchored to his back. By the time he reached his cabin he was exhausted. He stretched out on his bunk, drew his breath in sobbing gasps. No wonder colonization of Jupiter had proved so difficult. At length, he drove himself to his feet. The plan, which had been hatched in the head office of the T.I.S., would brook no delay. Captain Cabot would have to wait. Stooping, he pulled a bundle of tough, specially-treated fiberoid, a material used in the construction of space suits, from under his bunk, slung it across his shoulder. Next, he dragged forth a clock-like instrument to which had been attached a magnesium flare, and lastly a cylinder of hydrogen. The hydrogen Gavin had refined from water by a crude electrolysis. The rest of the equipment he had slipped from the engine room, working on it during leisure moments since the little death. He opened his door. The corridor was deserted. Twice during the ascent topside, Gavin had to stop and rest. Even breathing was an effort. At length he reached the arched outer skin of the monster, pried open an escape hatch. The thick yellow air of Jupiter poured down upon him like soup. It smelled and tasted faintly like swamp gas. He had a momentary fear that he would strangle. A spasm of coughing seized him as he gulped in the first breaths. Then, his lungs having adjusted themselves, he clambered to the outer shell. A hundred feet below, he could make out the surface, only sketchily visible through the yellow pea-soup air. Slave pens and cantonments
- 32. were all swallowed by the dense fog. He set to work assembling his instruments. The cold knifed to his bones. A wind was blowing, too. It pushed against him like the sluggish current of a river. The fiberoid package, unfolded, proved to be a balloon almost ten feet in diameter. It had a safety valve in it to neutralize the pressure when the bag reached the stratosphere. Gavin attached the clock and flare, started the clock in operation, inflated the bag. The instant it tugged at his numbed fingers, he shut off the hydrogen, cast it free. It was scarcely a fifth inflated, but the heavy pressure caused it to float slowly, upward out of sight. The clock was timed to ignite the flare when the balloon reached the stratosphere. Observatories on Io, Europa, Callisto, and Ganymede had their instruments trained on Jupiter. As soon as the flare was discovered, a check was to be made of the prevailing winds in that area. A simple parabola would indicate the balloon flare's probable course. The information then would be relayed to a flight of five patrol spacers held in readiness on the nearer moon. That was the way it had been planned. But now Gavin was not so sure. So many things could interfere. He closed the trap overhead and retreated back to his cabin. Sweating profusely, he flung himself on his bunk. He was still there, his breath rasping in his throat, when his door was pushed silently open from the outside. Gavin whipped his dart-gun from its spring clip and slipped it under his pillow. He didn't move, but lay still with his eyes closed except for the barest fraction of an inch. The door yawned wider. Then the figure of Nadia Petrovna slipped soundlessly inside, eased the door shut. She stood over him, watching him with a desperate intentness. Satisfied that he slept, she set to work searching his cabin.
- 33. Gavin lay quiet, observing her skill appreciatively. The girl was efficient. She went rapidly through his closet, his chest, his bags. She probed the lone chair cushion with a long needle, peered under his bed, then vanished in the shower. After a moment she reappeared, stood over him again, a puzzled expression on her pretty slavic features. "No luck, eh?" murmured Gavin pleasantly. He opened his eyes and sat up. Nadia gasped. Gavin narrowed his eyes and roared in a suddenly harsh voice, "What the hell are you prying through my luggage for?" The girl jumped. "I—I ..." she began, and then lapsed into confusion. He waited. "It sounds so foolish," she confessed, her long black eyes on the deck. Her fingers were twining about each other nervously. "But I can't get that dream out of my mind." She glanced up at him with a frightened expression. "What dream?" "The little death. When you turned out to be a T.I.S. agent. I—I begged you to give me a chance, let me try to escape." "Yes," asked Gavin with a show of interest. "What did I do?" "You laughed at me. I can't forget it." "I wouldn't laugh at you," he replied somberly. The girl's eyes softened. They were remarkable eyes, long and black and lustrous, the lashes half-hiding them. "Why, Gavin, I believe you're flirting with me." There was an odd little laugh in the words. It was the first time she'd called him Gavin.
- 34. "Don't be too sure," said Gavin gruffly. His arms went around her waist. He pulled her to a seat beside him and kissed her roughly. Someone rapped at the door. Nadia sprang to her feet. Gavin called out, "Who is it?" "The steward, sir. The Captain sent me to fetch you to the messroom." When Gavin entered the officer's mess, he saw the Captain seated across a table from the thinnest man he had ever encountered. "This is Hendricks," Cabot introduced them. The Captain was cold sober and hard-eyed. "He's the factor here at the cantonment." Gavin shook hands. The factor had an amazing grip. His flesh hung on in tough strings. He looked more like an animated skeleton than a man, but Gavin was conscious of a tremendous wiry strength in him. "You going ashore, Mr. Murdock?" the factor inquired. Gavin nodded. "Miss Petrovna and I were planning to visit the cantonment." He saw a glance pass between Cabot and the factor. The factor said, "Splendid," and rubbed his emaciated hands together. "Allow me to play host. The officers are already at my establishment." Gavin thanked him. "But let me warn you," went on the factor. "Don't wander off. The gravity aboard the ship here is bad enough. The atmosphere is much worse. It's easy to get lost, I've known newcomers to die of exhaustion only a few steps from the cantonment." Gavin thanked him again, started to withdraw when the factor halted him. "I've bearers waiting outside," he explained. "They're at your service while you're here. I really advise you not to walk any more than
- 35. necessary." This time Gavin made good his escape. He had been politely instructed not to pry, he realized. Villanowski might be confident of him, but not the Captain. Gavin was smiling when he met Nadia at the main port, but his eyes were hard. Both of them had donned outer insulating garments of thermal cloth. "We're going to be carried in litters like ancient Oriental potentates," he informed her. Nadia laughed. "You've never been to Jupiter before?" He shook his head. They passed through the lock into the swirling yellow air. It was like swimming. They crept down the gangplank. The bearers were standing patiently at the surface. Gavin had seen Jovian dawn men before, but they never failed to excite his curiosity. Huge, almost seven feet tall, and muscled like gladiators, they were imposing as Greek gods. Their skin was the vivid blue of polished turquoise, their long manes as yellow as a sunburst. "There's a double litter," Nadia pointed out. "Let's take that." Gavin followed her inside. The litter was cushioned like a divan. One was borne along in a semi reclining position. Nadia clapped her hands and cried, "Cantonment!" Four naked blue giants swung the litter to their shoulders and started off at a rapid trot. In a few paces, the ship had disappeared. They were like a tiny raft, alone in a welter of yellow oppressive fog. Gavin, peering over the edge, saw that the giants were following a paved road. After a moment they began to pass an endless procession of dawn men, chained ankle to ankle and moving sluggishly toward the Nova. Already the loading had started.
- 36. Gavin stared at the half-men curiously. They crept along, features drawn, their yellow manes matted with dirt. There was a haunted look in their eyes like caged animals. Thirty percent would die in the crossing, he knew. Within a year ninety percent of the rest would be dead, victims of home-sickness, of pneumonia and measles and Venusian lung rot, not to mention a score of other diseases. The terrible rate of fatality was good business for the slavers. It held up the demand. "They aren't really human," said Nadia in a faint voice as if reading his thoughts. "They're sub-men." Gavin nodded. Some place along the evolutionary scale the Jovians had taken a wrong turning. They would never evolve into true homo sapiens. But even cattle weren't treated as they were. The walls of the cantonment hove suddenly in view through the murky atmosphere. The giants paused before a massive entrance like the gate of a feudal castle. Gavin climbed out of the litter and started to help Nadia down. Somewhere off in the distance he heard a faint popping. "What is it?" cried the girl, struck by his strained air of attention. "It sounds like the dum-dum fire. Listen!" They both fell silent. From the direction of the Nova came faintly a sound like a string of fire-crackers going off together. "It is dum-dum fire!" "But it can't be! It's impossible." The sound of explosions drifted to them again. Gavin sprang back into the litter.
- 37. "Wait here," he cried. "Something's wrong at the ship." He knew that it couldn't be the Terran patrol spacers attacking. The balloon hadn't time to rise into the stratosphere yet. A great fear for the Nova's safety gripped him. Whatever the cost, the ship must be preserved intact so that Terra's scientists could examine her space drive. It had become the paramount issue, over- shadowing in importance even the detestable slave trade. "No!" Nadia cried. "Don't go back there." She flung herself on him, pressing her body flat against his. "Please, for my sake, Gavin!" He pushed her rudely aside. Just then the chief astrogator burst upon them, running from inside the cantonment. His strides were labored, his breath wheezing in his chest. He saw Gavin and shouted: "The ship's being attacked! They've radioed the cantonment for help!" "Who by?" "X's men!" The chief astrogator began to run down the paved highway toward the Nova and was swallowed by the fog. Gavin heard something whine through the air. He flung himself flat on his face, shouting at Nadia, "Down! They're shelling the cantonment!" The shell burst out of sight in the fog. A blast of air hit him like a wall of water. Nadia sat up, her face smudged where she'd groveled in the dirt. She was cursing like a spaceman. Gavin yanked her to her feet. "Clear out!" He began to haul her away from the doomed cantonment. Another shell lobbed over their heads to explode directly behind them. Gavin's hands were torn from the girl. He was blown a dozen feet by the blast. He lay where he lit, knowing nothing, feeling nothing.
- 38. VIII Gavin's first impression was one of numbing cold. He opened his eyes. Pitch blackness engulfed him. He had difficulty orienting himself. Rather hazily, he recalled the shelling of the cantonment by X's men. Gradually his mind cleared. The swift Jovian night had fallen, he perceived, and the temperature had dropped sharply. Only his insulating outer garments had saved him from freezing. He began to grope around for Nadia's body. He found nothing but bare ground, stones, shrubs. He sat back on his haunches, getting his bearings. The night pressed against his eyeballs; silence rang in his ears. No popping of dum-dum fire was to be heard. Whether the raiders had won, or Cabot was still in possession of his ship, he couldn't tell. But the fight was over. X, himself, he realized, couldn't possibly have reached Jupiter yet. Even if he had been aboard one of the ships which had pursued the Nova out from Venus, a whole year must elapse before he could arrive. Then, if X's men had attacked the Nova, they had been planted here earlier and had been waiting in ambush. That meant X had been tipped off to the location of the hidden slave cantonment on Jupiter. Something brushed against Gavin's face! It felt like cold fingertips. Gavin's arms flailed the air in stark terror. He struck a soft, cold, hairless body. There was a barrage of half-human squeaks. The air was full of the fluttering of wings. Scavenger bats! Gavin felt a prickling of cold sweat break through his skin. He began to grope feverishly for the girl's body again, working outward in an ever-widening spiral.
- 39. After an hour he had lengthened the radius of his search until he was among the debris of the cantonment. He sat back on his haunches, sure of only one thing. Nadia Petrovna was not there! The air above the demolished cantonment was thick with the squeaks and wing rushing of the hairless bats. A faint yellow glow heralded the dawn. If the Nova hadn't sailed, there was still hope. Gavin drove himself to his feet, prowled the debris in search of the road. He started hundreds of the half-human scavenger bats into whispering, squeaking flight, stumbled across countless bodies. The raiders had been thorough. At length he found the paved highway, began to follow it by feel. A wind was blowing across the road. Gavin had to fight it like a man fording a stream against a strong current. The light, though, continued to brighten until he could make out the trace beneath his feet. Then the towering bulk of the Nova loomed dead ahead. She wasn't gone! Gavin flung himself gasping on the ground, trembling with exhaustion. He rested only long enough to control his trembling muscles, then began to skirt the ship towards the blind spot in the rear. He stayed out of sight. He had no desire to be spied by a possible watch posted at the scanner. The disposal chute was just forward of the rear jets. Gavin reached it unobserved, as far as he could see, and began to worm his way up the inside of the tube like a climber ascending a chimney. He reached the lock and got his shoulder beneath it. The lock had been designed to operate in space where the pressure inside the ship helped seal it. Now, aided by Jupiter's dense atmosphere, he succeeded in prying the lock up and scrambling into the trap. Ten minutes later, he climbed out of the chute directly aft of the galley. The air was warm and light, bringing him the odor of cooking soup.
- 40. The passage was deserted. Gavin slipped into the escape well which led from the engine room to the Nova's outer skin, clambered downward again. He stepped from the escape well softly into the engine room itself. At first he thought it was deserted. Then he discovered a guard posted beside the sealed door in the aft bulkhead. Gavin had never seen him before. He was a big Terran in coarse outer garments. He was facing half away from the T.I.S. agent, holding a dart-gun. Gavin slipped his fingers through his brass knucks. He edged cautiously from behind the Nova's cyclotron, crept up on the man with the stealth of a ferret. At the last moment, the fellow heard him and swung around. Gavin clipped him behind his ear with the weight of his shoulder back of the blow. The guard's head banged against the steel bulkhead. He slipped nervelessly to a sitting posture, tumbled sideways. His breath bubbled with a rattling sound from his mouth. Then he stopped breathing. Without bothering to check his pulse, Gavin turned to the control panel. The fuel gauge showed a comfortable surplus. Ears straining to catch any untoward sound, he slowly pulled down the lever which dumped the fuel, watched the gauge with a growing tensity of nerves. The level in the gauge dropped tantalizingly slowly, as the liquid fuel bubbled out of the tanks onto the surface of Jupiter to saturate the soil. It was still a half-inch from the empty symbol when he heard voices. Someone was descending the ladder into the engine room. Gavin's jaw set; his lips thinned. With his left hand he drew his dart- gun, but he didn't release the dumping lever. The gauge showed a
- 41. three-eighths of an inch, then a quarter. A pair of boots descended into his range of vision followed by the legs and waist of a man. The man reached the deck, faced around and stared at the T.I.S. agent in astonishment. "Don't move!" began Gavin. Another voice from up the ladder barked "Drop that gun!" Gavin's eyes flashed upward. He saw a man's head, shoulder and arm through the circular ladder well. The man seemed to be lying on the deck above covering him through the opening with a dart-gun. Gavin dropped his own automatic. "Take your hand off that lever!" the man snapped. Gavin flicked his eye to the gauge. The last of the fuel was flowing from the tanks. He released the lever, straightened his shoulders. Let them do what they pleased to him now, he thought, they were too late. The Nova was grounded! The second man descended the ladder and the pair of them regarded him curiously. They were both big men, Terrans in baggy outer garments like the guard whose skull Gavin had cracked. "Who the hell are you?" asked the first man in a flat voice. These men were bad. They didn't play at it. They didn't try to be. It was etched in their cold eyes and tight mouths. Gavin moistened his lips. "Murdock. Third assistant-engineer." "Another one," exclaimed the second man in faint surprise. "I thought we'd bagged the lot." The other grunted. "We'd better take him to Y." Gavin was searched and then hustled up the ladder into the officer's mess. As he was propelled through the door, conversation died in the messroom, and four pairs of eyes turned on him curiously. Gavin controlled his surprise. Villanowski was there, ironed to his chair, his homely features taut with strain. At the table to the left of Villanowski sat the emaciated factor. He wasn't ironed. Neither was
- 42. Nadia Petrovna. She had changed into crisp shorts and was leaning forward, lips parted in surprise. But it was the fourth man who drew the T.I.S. agent's attention. He sat between Nadia Petrovna and the factor, lolling back in his chair indolently, a sheaf of papers spread on the table before him. His face was like a death mask in which the coloring, the lines had been painted by a machine. It was perfect, but without life. Then Gavin realized that it was a mask. The man's whole face was a lie, even to the realistic mole on his chin. He leaned across the table. "Who's this?" he asked in a strong harsh voice. "He says his name's Murdock. Claims he was third assistant-engineer. We found him down in the engine room. He's killed Peters and dumped all the fuel." Nadia drew in her breath sharply, looked frightened. The factor's fleshless features blackened with rage. Even Villanowski glanced up, a look of surprise and dawning hope in his eyes. Only the man in the mask didn't change expression. "Go back to your post," he ordered Gavin's captors. "We'll take care of Mr. Murdock." Then, when they had left, he said, "You were contacted by my—ah—co-worker on Venus, Murdock. Mr. X, he called himself." He chuckled, the noise issuing from between unsmiling painted lips like the voice of an automaton. "For the sake of convenience, you can term me Mr. Y." Gavin didn't say anything. "You have presented us with a problem," Mr. Y went on. "Where's Cabot?" Gavin interrupted. "Dead." "The chief astrogator, the crew?" "All dead. In fact, we thought we'd wiped the slate clean."
- 43. The factor suddenly slammed the table with his bony fist. "What are you playing with him for, Y? I've sacrificed everything. The cantonment, the slaves. I demand that he be done away with before he can contrive any more damage." Y regarded the factor with venomous amber eyes, the only living features in his death mask. "Has it occurred to you to wonder why Mr. Murdock dumped the Nova's fuel?" The factor started. "Mr. Murdock," said Y, turning back to Gavin, "what persuaded you to ground the Nova?" Gavin's lips thinned. He didn't say anything. Y continued to regard him a moment. Then he asked Nadia, "I believe you suspected Murdock of being an agent of United Spaceways, Miss Petrovna?" Before the girl could answer the factor broke in again. "Why can't we use the Nova's special space drive?" "You're referring to the machinery of the little death," rejoined Y. "But I thought you knew. It can't operate until the Nova has attained a certain velocity. That much we've ascertained from Mr. Villanowski." "Then we're trapped!" The factor leaped to his feet. His agility in the increased gravity was amazing. Gavin realized that the long years the factor had spent on Jupiter had trained his muscles as well as wrung every ounce of extra flesh from his spare frame. "I see that you've comprehended our position at last," said Y grimly. "What about the emergency fuel tanks at the cantonment?" "Gone. Your damned shelling exploded them." Y nodded. "Just as I thought. If Murdock drained the Nova's tanks, he must be expecting help. It will have considerable bearing on any course we plan to take, just who these aids are, how many they'll be, and when to expect them."
- 44. He returned to Gavin. "You'll spare yourself a painful experience, Murdock, if you talk now. You can't gain anything by forcing us to wring the information from you. We won't hesitate to stoop to torture." "No," Gavin agreed. "I suppose not." He hesitated. United Spaceways and Tri-World were the two corporations most likely to want the Nova's space drive. Nadia suspected that he was an agent of United Spaceways. Therefore ... he moistened his lips. "My work's done, anyway. I'm a Tri-World agent." "But that's impossible!" Nadia burst out in sudden protest. "We...." She paused, looking confused as she realized she'd been tricked. Y said, "That was very clever, Murdock. Yes. I'm working for Tri- World. Miss Petrovna and my good friend, the factor, have supplied us with valuable information and help for a price. But the knowledge won't be of any earthly use to you." Gavin felt no triumph at the confirmation of his suspicions. What Y said was true. As soon as they had squeezed him dry, he would be silenced. The door opened. A Terran appeared. "The slaves have all been unloaded and dispersed, sir. The bodies of the crew are buried. We're ship-shape and ready to take off." Y put a forefinger to the chin of his death mask, rubbed the plastic gently. "Establish radio contact with the Comet." "Yes sir," replied the man. "The message, sir?" "Rendezvous cancelled. Emergency. Proceed to us at once prepared to divide fuel." "Yes, sir." The man went out, closing the door softly behind him. IX
- 45. Gavin's heart sank. Of course the ship that brought Y would be hidden in the neighborhood. It would be simple enough for them to refuel the Nova. Y had taken time to release the slaves and dispose of the bodies of the crew. That, Gavin surmised, was what had delayed the Nova's take-off long enough for him to slip aboard. Now should the Nova be apprehended in space, the Interplanetary patrol would be forced to release her for lack of evidence. Villanowski glanced up. "We're licked, lad. We may as well toss in the towel." Gavin looked at the chief engineer blankly. "You mean," asked Y of Villanowski, "that you and Murdock are working for the same organization?" The chief engineer laughed bitterly. "We're not working for anyone, but ourselves. You forget, Mr. Y, that the four-dimensional drive isn't the only contribution I've made to interplanetary travel. General Atomic stole the others. We had hoped—" he jerked his head at Gavin "—to keep this one for ourselves!" Gavin's mind was going in circles like a dog chasing its tail. What was Villanowski's motive? "Go on," said Y in a foreboding voice. Villanowski looked down at his chains. "A ship travels through space during a passage of time. It had occurred to me that if I could invert the formula and drive a ship through time during a passage of space, the crude rocket ships could be abandoned. Murdock had gotten in trouble when Transplanet was discovered to be a colonal smuggling ring. He had studied astrophysics under me at New Yale. I knew him and knew I could trust him." Gavin swallowed and struggled to keep a grip on himself. Obviously Villanowski had something up his sleeve. Villanowski looked at Gavin. "We needed a space ship to complete our experiments. The effect of the drive on a body at rest was
- 46. startling enough to predict success if we could attain sufficient velocity." Gavin thought he detected a faint stressing of the word "startling". Villanowski had said, "The effect of the drive on a body at rest was startling...." The Nova was at rest! "I persuaded Cabot," Villanowski proceeded, "to let me install the mechanism aboard the Nova. Murdock was to try—" Gavin jumped. With a back-handed, edge-on swipe, he caught Y in the throat full on his larynx. Nadia screamed as Y went over backwards and lay still. The factor leaped to his feet. Gavin kicked him in the belly. Spinning against the girl, he wrenched out of her hand the dart-gun which she was drawing. "The keys!" Villanowski panted. "Y has them." Nadia opened her mouth to scream again. "Don't." Gavin pointed the dart-gun straight at her open mouth. Nadia shut it. The factor was writhing on the deck, but Y lay like dead. Gavin found the keys and released Villanowski. "The engine room," he cried, "we've got to reach the engine room." "Take Y's gun," said Gavin. He turned on Nadia. "Come along. We can't leave you here to sound an alarm." Nadia's lips were bloodless. She moved stiffly between them, Gavin's dart-gun prodding her gently in the spine. They reached the engine room without being discovered and disarmed the startled guard. Villanowski whistled a bar in C sharp and then said, "Open sesame." The door in the steel bulkhead swung soundlessly outward.
- 47. There was a faint grin on Villanowski's homely face. "Sound waves set its mechanism in operation. I read a story when I was a youngster—Ali Baba and the Forty Thieves," he confessed. "When I built the lock I couldn't resist designing it to respond to those vibrations." The ship rocked slightly. "What's that?" "Felt like the blow-back of an atom jet," Gavin replied tersely. "I think Y's ship, the Comet, is landing." The Nova rocked again, more violently than before, and a faint rush of flaming jets penetrated to the engine room. Villanowski scuttled through the door with Gavin prodding Nadia after him. The chief darted for the control board, seized a gleaming lever, slid it cautiously along a slot. The huge spherical tube, which Gavin had observed before, began to glow gently. The yellow mist, he saw, was gathering in the outer room, but the air in their chamber remained crystal clear. "The time field," Villanowski explained, "creates a neutral area, an oasis, around the point of generation." Gavin rubbed his eyes. On the other side of the door was nothing! It was like looking out into the void beyond the farthermost limits of the stars. "Now, Murdock," Gavin heard Villanowski ask, "when do you expect the interplanetary patrol spacers to land?" Gavin wheeled around. There was an uncertain smile on Villanowski's homely features. "How did you know?"
- 48. "The little death," Villanowski explained complacently. "When Miss Petrovna told me her dream, I knew...." "You are a T.I.S. agent!" Nadia interrupted in an odd voice. She put her hand on Gavin's sleeve. "You're going to turn me over to the courts?" Two white patches appeared at the corners of Gavin's mouth. "Sure. The patrol spacers are on their way. I couldn't turn them back now—" he paused—"even if I wanted to." "Give me a chance, Gavin?" He asked, "Why did you sell out to Tri-World?" The girl lifted her eyes to his. "I was proscribed. Tri-World has granted me amnesty. That was their price. My life. I wouldn't have done it, Gavin, but I was afraid." "Does that explain why you tried to double-cross Tri-World in the first place?" "I told you about that." "And what about Trev? He hid you out when you were proscribed. He got you aboard the Nova. Why did you sell him out to Cabot?" "But I didn't. I—I...." "Don't lie to me," he said and gripped her shoulders. "You'd made your dicker with Tri-World. Trev was in your way. He knew too much about you. He might even have got wind of what you were up to. So you went to Cabot and told him about Trev being a dealer in scientific secrets. You knew Cabot would kill him, thinking the Martian had sold out to X." The girl flinched. "I'm not asking you to shield me, Gavin. Turn me loose. Just let me have a fighting chance to escape. Give me a chance, Gavin." "And Cabot," Gavin continued inexorably, his pale blue eyes stony. "You delivered Cabot and the Nova over to Tri-World."
- 49. He turned her loose. "Give you a chance," he repeated and gave a short bitter laugh. "A chance to do what? Double-cross me like you have everyone else?" Nadia shrank away. "The dream!" she said in a frightened voice. "It's just as it was in the dream. You laughed!" Villanowski interrupted sadly. "You weren't dreaming during the little death. We're only equipped with three dimensional sense organs. We're blind to everything but the immediate instant. But time's a dimension. It's co-existent. When the Nova was projected across time, your entire life was spread out around you. What you actually did was experience a segment of your life. It happened to be a segment in the future." Nadia's lips were bloodless. "You guessed when I told you about the dream?" "I didn't guess," replied Villanowski. "I knew! Miss Petrovna, if you saw the Nova captured by the Interplanetary Patrol through efforts of Murdock who was a T.I.S. agent, then it was inevitable that it would take place exactly as you had seen it. There was nothing any of us could do about it!" A faint grin broke across Villanowski's homely face. "I saw that it behooved me to give Murdock a hand when and if he needed it." Gavin said, "I think I can promise you amnesty, Villanowski. I couldn't have captured the Nova without your help." "Oh, that's not all," Villanowski chuckled. "The Empire will want this space drive to power her ships. I'll be a valuable man. Even Y didn't intend to kill me until I had explained its mechanism to Tri-World's scientists." "How long," asked Gavin abruptly, "have we been on Jupiter? I lost track after that shell knocked me out at the cantonment." "This is the second day."
- 50. Gavin wrinkled his brow. "The patrol ships should be here in about four hours." Outside the Nova, a gaping hole in space marked her position. The astounded crew of the Comet, who had landed prepared to refuel the captured Nova, eyed the eerie vacancy with mixed emotions. One of the crew flung a rock into the enveloping blackness. It disappeared. There was no sound of its falling to the ground. The commander of the Comet, deprived of Y's guidance, fumed nervously. He glanced at his watch at intervals, saying at length, "If the Nova doesn't reappear by dark, we'll take off. We can lay up and re-establish contact by radio." The men gathered about the maw of blackness staring into it with hypnotic fascination. They fell an easy prey to the five sleek patrol craft which plummeted down on them three hours later. The short Jovian day was on the wane when the Nova began to gather substance like a tenuous cloud. Her misty outlines grew solid. Then the port opened. Gavin Murdock appeared in the entrance. Commandant Samuels, a grizzled veteran of the T.I.S., was the first man up the gangplank. He shook Gavin's hand. "Nice work, Murdock. But it smacks damnably of witchcraft." The Flight Commander was right behind him, followed by the captains of the Empire's patrol spacers. The Flight Commander caught sight of two of Y's men lying unconscious just within the port. "What the hell's this? The palace of the sleeping beauty?" "You haven't seen the half of it," Gavin assured him with a grin. "They're lying all over the ship like that. Villanowski says the effects last about an hour. Better lock them up before they come to." "Villanowski?" echoed the T.I.S. Commandant. "He's not dead, is he?"
- 51. "No," said Gavin, surprised at the anxiety in the Commandant's voice. "Good!" growled Samuels. He lowered his voice to a subdued roar. "Ticklish mission. I'm supposed to persuade him to accept a post on the Empire's Bureau of Research. They're afraid his space drive will fall into wrong hands. But Villanowski's such an embittered old goat, he'll probably...." There was a faint chuckle behind Gavin. Villanowski, who had come up unobserved, said, "Your diplomacy, Commandant Samuels, is unique." There was a broad grin on his homely face. "I couldn't think of opposing such finesse." The grizzled T.I.S. Commandant's features flamed an apoplectic red. Then he burst into laughter, wiped his eyes, and blew his nose. "Where's Nadia?" Gavin asked sharply. "Ironed to my old chair in the officer's mess." Villanowski handed Gavin the keys. Gavin passed them on to the Commandant. He gave a terse but concise report, while the men filed aboard and began to cart the unconscious Tri-World agents off to the patrol spacers. Y was found to be dead, the blow on his esophagus having killed him. When the mask was stripped from his face, Commandant Samuels identified him in amazement as the chief of Tri-World's gestapo. "We caught X, too," he said. "We received a flash aboard the flagship that X has been captured in space." "What was he charged with?" "Piracy. Y's men will be tried on the same charges. Tri-World, of course, will disclaim any connection, but she'll have to rebuild her gestapo from top to bottom."
- 52. Gavin moved aside to allow two men with a stretcher to pass out the lock. The body of the factor lay on the stretcher looking thin as a straw. He was alive, Gavin noted, seeing his chest move faintly. "This gives the death blow to the slave trade," the Commandant began. Then he realized Gavin wasn't listening. Nadia Petrovna followed the stretcher. Her hands were in irons and a guard walked beside her. She passed silently between the men, her black eyes flashing Gavin a look of hate before she descended the gangplank. "Mark my word," said Commandant Samuels grumpily, "that girl will get off with a light sentence. She'll run true to form and sell Tri-World down the river. She'll be the prosecution's principal witness." Gavin shrugged. "Which reminds me," put in Villanowski. "Since the Empire's so anxious for me to return to the fold, I don't want to appear too eager." "Eh?" A pained expression rippled the T.I.S. Commandant's ruddy features. "I'd like the chief engineer's rating aboard the first ship to reach the stars." There was a wistful note in Villanowski's voice. "There shouldn't be any hitch there," the Commandant agreed in a relieved tone. "You're the logical man to head an expedition outside the system." "What about a third assistant-engineer?" Gavin interrupted. "We work pretty well together," said Villanowski. Gavin's lean freckled face broke into a grin. "It's a bargain." The two men solemnly shook hands.
- 53. [Transcriber's Note: Original text had two Section VIII headings. Second heading renumbered to IX.]
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