Microarray
- 2. DNA Microarray DNA microarrays are microscope slides that are printed with thousands of tiny spots in defined positions, with each spot containing a known DNA sequence or gene. These slides are referred to as gene chips or DNA chips. DNA microarrays are solid supports, usually of glass or silicon, upon which DNA is attached in an organized pre-determined grid fashion. Each spot of DNA, called a probe, represents a single gene. A microarray is a laboratory tool used to detect the expression of thousands of genes at the same time.
- 4. A basic protocol for a DNA microarray is as follows: Isolate and purify mRNA from samples of interest. Since we are interested in comparing gene expression, one sample usually serves as control, and another sample would be the experiment (healthy vs. disease, etc) Reverse transcribe and label the mRNA. In order to detect the transcripts by hybridization, they need to be labeled, and because starting material maybe limited, an amplification step is also used. Hybridize the labeled target to the microarray. This step involves placing labeled cDNAs onto a DNA microarray where it will hybridize to their synthetic complementary DNA probes attached on the microarray. A series of washes are used to remove non-bound sequences. Scan the microarray and quantitate the signal. The fluorescent tags on bound cDNA are excited by a laser and the fluorescently labeled target sequences that bind to a probe generate a signal. The total strength of the signal depends upon the amount of target sample binding to the probes present on that spot
- 6. cDNA slide fabrication mRNA preparation, fluorescence dye labeling, gene hybridization robotic spotting green and red fluorophores excitation by lasers imaging using optics slide scanning analog to digital conversion using image storage and archiving. GENRAL cDNA MICROARRAY WORKFLOW
- 7. Parameters affecting fabrication of microarrays. Spot density and array geometry. Probe density is defined as number of probes located on a given surface while hybridised density is defined as the number of target located on a given surface Hybridisation efficiency is defined as the ratio between hybridised and probe density
- 8. Classification of microarray based on the mode of preparation 1. The spotted array on glass: spotted arrays are arrays made on poly-lysine coated glass microscope slides. This provides binding of high-density DNA by using slotted pins. It allows fluorescent labeling of the sample. 2. Self-assembled arrays: these are fiber optic arrays made by the deposition of DNA synthesized on small polystyrene beads. The beads are deposited on the etched ends of the array. Different DNA can be synthesized on different beads and applying a mixture of beads to the fiber optic cable will make a randomly assembled array. 3. In-situ synthesized arrays: these arrays are made by chemical synthesis on a solid substrate. In the chemical synthesis, photolabile protecting groups are combined with photolithography to perform the action. These arrays are used in expression analysis, genotyping, and sequencing. Affymetrix for instance produces microarrays routinely with millions of probes on 1.28 cm2 surface. There is almost no space between the spots and spotsizes are below 10 mm.
- 11. (A) Unmodified DNA is randomly immobilised to surfaces meaning that some DNA strands can participate in hybridisation (I) while other cannot (II). (B) Immobilisation of DNA using end modifications (I) can also result in intra chain bonds (II). (C) Molecular organisation of end modified probes directly immobilised to the active groups on the solid support (I) or displaced from the surface using a linker (II) or a dendrimeric linker (III). Immobilisation of DNA to surfaces.
- 14. A grid alignment (also known as addressing or spot finding or gridding) is one of the processing steps in microarray image analysis that registers a set of unevenly spaced, parallel, and perpendicular lines (a template) with the image content representing a two-dimensional (2D) array of spots. Grid alignment methods: Manual grid alignment, semiautomated grid alignment and fully automated grid alignment. Grid alignment
- 15. Foreground and background detection. The outcome of grid alignment is an approximation of spot locations. The next task is to identify pixels that belong to foreground (signal) of expected spot shape and to background. This task involves image segmentation and clustering. Image segmentation is associated with the problem of partitioning an image into spatially contiguous regions with similar properties (e.g., color or texture) image clustering refers to the problem of partitioning an image into sets of pixels with similar properties (e.g., intensity, color, or texture) but not necessarily connected.
- 16. (1) circular signal area outside of allowed location and radius deviations, (2) small signal with respect to background (3) disconnected signal areas (4) inconsistent intensity probability distributions. quality assurance of the microaray image is the process of elimination of the grid cells with unreliable microarray information. This is done by running the screening algorithm which performs four types of quality assurance. Quality assurance Normalization of microarray data is aimed to correct for the systematic measurement errors and bias in the observed data. The errors and bias may be introduced by several factors such as difference in probe labeling, concentration of target DNA/ RNA sequence, efficiency of hybridization, instrumental noise due to scanners or printers etc.
- 17. Classification of microarray based on the types of probes used 1. DNA microarrays: DNA microarray is also known as gene chip, DNA chip, or biochip. It either measures DNA or uses DNA as a part of its detection system. There are four different types of DNA microarrays: cDNA microarrays oligo DNA microarrays – In this type, the spotted probes contains of short, chemically synthesized sequences, 20-25 mers/gene. specificity is high and cross hybridization can be inhibited. BAC microarrays - A BAC clone chip is a DNA microarray whose probe is a template amplified by PCR. The template is a genome region incorporated into a comprehensive BAC (bacterial artificial chromosome) clone which various research institutions used in decoding the genome sequence of various organisms. SNP microarrays. - SNP array is a type of DNA microarray which is used to detect polymorphisms within a population
- 19. Analytical microarrays Analytical microarrays are typically used to profile a complex mixture of proteins in order to measure binding affinities, specificities, and protein expression levels of the proteins in the mixture. Antibody microarrays are the most common analytical microarray In this technique, a library of antibodies, aptamers, or affibodies is arrayed on a glass microscope slide. The array is then probed with a protein solution.
- 21. Functional Microarrays functional protein arrays are composed of arrays containing full-length functional proteins or protein domains. These protein chips are used to study the biochemical activities of an entire proteome in a single experiment. They are used to study numerous protein interactions, such as protein–protein, protein–DNA, protein–RNA, protein– phospholipid, and protein–small molecule interactions
- 23. In RPA, cells are isolated from various tissues of interest and are lysed. The lysate is arrayed onto a nitrocellulose slide using a contact pin microarrayer. The slides are then probed with antibodies against the target protein of interest, and the antibodies are typically detected with chemiluminescent, fluorescent, or colorimetric assays Several such multiprotein samples are spotted on the microarray, which is then probed with a single target molecule. Reverse Phase Microarrays
- 25. An ideal surface for protein microarray fabrication has to be capable of immobilizing proteins and preserving their three‐dimensional (3‐D) conformation.
- 27. 3. Tissue microarrays: tissue microarray paraffin blocks that are formed by separating cylindrical tissue cores from various donors and embedding it into a single microarray. This is mainly used in pathology. 4. Cellular microarrays: they are also called transfection microarrays or living- cell-microarrays, and are used for screening large-scale chemical and genomic libraries and systematically investigating the local cellular microenvironment. 5. Chemical compound microarrays: this is used for drug screening and drug discovery. This microarray has the capacity to identify and evaluate small molecules and so it is more useful than the other technologies used in the pharmaceutical industry. 6. Carbohydrate arrays: they are also called glycoarrays. Carbohydrate arrays are used in screening proteomes that are carbohydrate binding. They can also be utilized in calculating protein binding affinities and automization of solid- support synthesis for glycans. 7. Phenotype microarrays: phenotype microarrays or PMs are mainly used in drug development. They quantitatively measure thousands of cellular phenotypes all at once. It is also used in functional genomics and toxicological testing.