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Microarrays: Tools & Interpretation of Data Kirsty Jensen Microarrays • What is a microarray? – How the technology has evolved • Macroarrays • Glass microarrays • Affymetrix • Designing a microarray experiment – Choice of arrays – Experimental design – RNA samples • Analysis – – – – – – Scanning Quantification of spots Normalization Statistical analysis Identification of genes of interest Validation What Is A Microarray? “A microarray is a tool for analyzing gene expression that consists of a small membrane or glass slide containing samples of many genes arranged in a regular pattern.” Uses • • • • • Analysis of gene expression SNP analysis MHC typing Identification of pathogens Identification of tumours Microarrays • The underlying principle of microarray technology is the ability of DNA to bind to itself and to RNA. • Analyzing gene expression involves the detection of mRNA species (transcriptome) present in a cell or tissue at a particular point in time. Evolution of Microarrays • One gene / one experiment – Northern blots, RNase assays, RT-PCR – Hypothesis driven • Macroarrays – Nylon membranes – 32P labelled probes Macroarrays RNA RNA 32P labelled 32P labelled cDNA cDNA Hybridization Hybridization Evolution of Microarrays • One gene / one experiment – RT-PCR, northern blots, RNase assays – Hypothesis driven • Macroarrays – Nylon membranes – 32P labelled samples • Microarrays – Glass slides – Fluorescent labelled samples – Two samples per microarray Microarrays RNA RNA Cy3 labelled cDNA Cy5 labelled cDNA Hybridization Glass Microarrays • cDNA arrays – >1000 nucleotides in length – cDNA libraries • Oligonucleotide arrays – 70-80 nucleotides in length – Increased specificity Evolution of Microarrays • One gene / one experiment – RT-PCR, northern blots, RNase assays – Hypothesis driven • Macroarrays – Nylon membranes – 32P labelled samples • Microarrays – Glass slides – Fluorescent labelled samples • Affymetrix Affymetrix • 25 nucleotides in length • Mismatch pair – Single mismatch at centre of probe – Used to detect & eliminate false results • 11 probe pairs per gene • Photolithography Affymetrix - Construction Affymetrix Affymetrix RNA cDNA Biotin-cRNA Hybridization Streptavidin-phycoerythrin Bovine Microarrays Microarray Format Source of Material BOTL cDNA Bovine total leukocyte cDNA library & immune gene amplicons Pyxis CattleArray 7600 cDNA Bovine spleen & placental cDNA libraries NBFGC cDNA MARC & BARC cDNA libraries BMAM cDNA MARC & BARC cDNA libraries Mammary gland specific Endometrial/placental microarray cDNA Bovine placental & endometrial cDNA libraries Bovine immuneendocrine microarray cDNA Bovine immune & endocrine gene amplicons Bovine MHC class I microarray oligos BoLA DRB3 & DQA sequences BoMP microarray RIGUA cDNA Oligos Bovine macrophage cDNA library Bovine immune & inflammatory gene amplicons Affymetrix oligos Various cDNA libraries ARK-Genomics cDNA Various cDNA libraries Conclusions (1) 1. Microarrays have many uses. 2. Major use is to investigate gene expression. 3. mRNA isolated from a cell gives a snapshot of the events occurring at that time. 4. There are 3 main types of microarrays • Glass cDNA microarrays • Glass oligonucleotide microarrays • Affymetrix