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Genomics I: The Transcriptome RNA Expression Analysis Determining genomewide RNA expression levels Genomewide expression analysis • Goal: to measure RNA levels of all genes in genome • RNA levels vary with the following: – Cell type – Developmental stage – External stimuli • Time and location of expression provide useful information as to gene function Genomics expression analysis methods • Microarrays – Hybridization based • RNA-seq – Direct sequencing of cDNAs • SAGE (Serial Analysis of Gene Expression) – Sequence fragments of cDNAs • Real-time PCR Macroarray Analysis Macroarray Analysis Microarray Analysis of Transcription Animation Northern blots vs. microarrays target – • Global expression analysis: Northern blot – Limited by number of probes that can be used simultaneously • Global expression analysis: microarrays – RNA levels of every gene in the genome analyzed in parallel loading – control Basics of microarrays • DNA attached to solid support – Glass, plastic, or nylon • RNA is labeled – Usually indirectly • Bound DNA is the probe – Labeled RNA is the “target” Microarray hybridization samples • Usually comparative – Ratio between two samples • Examples – Tumor vs. normal tissue – Drug treatment vs. no treatment – Embryo vs. adult mRNA cDNA DNA microarray Two major types of microarrays • cDNA arrays- PCR product corresponding to a portion of a cDNA is immobilized on the slide • oligonucleotide arrays- oligonucleotide complementary to transcript is synthesized on slide or immobilized on the slide How microarrays are made: spotted microarrays • DNA mechanically placed on glass slide • Need to deliver nanoliter to picoliter volumes – Too small for normal pipetting devices • Robot “prints,” or “spots,” DNA in specific places DNA spotting I • DNA spotting usually uses multiple pins • DNA in microtiter plate • DNA usually PCR amplified • Oligonucleotides can also be spotted DNA spotting II • Pins dip into DNA solution in microtiter wells • Robot moves pins with DNA to slides • Robot “prints” DNA onto slide – DNA sticks to slide by hydrostatic interactions • Same spots usually printed at different locations – Serves as internal control • Pins washed between printing rounds • Hundreds of slides can be printed in a day Commercial DNA spotter How microarrays are made: Affymetrix GeneChips • Oligonucleotides synthesized on silicon chip – One base at a time • Uses process of photolithography – Developed for printing computer circuits Affymetrix GeneChips • Oligonucleotides – Usually 20–25 bases in length – 10–20 different oligonucleotides for each gene • Oligonucleotides for each gene selected by computer program to be the following: – Unique in genome – Nonoverlapping • Composition based on design rules • Empirically derived Photolithography • Light-activated chemical reaction – For addition of bases to growing oligonucleotide • Custom masks – Prevent light from reaching spots where bases not wanted • Mirrors also used – NimbleGen™ uses this approach lamp mask chip Example: building oligonucleotides by photolithography light • Want to add nucleotide G • Mask all other spots on chip • Light shines only where addition of G is desired • G added and reacts • Now G is on subset of oligonucleotides Example: adding a second base • Want to add T • New mask covers spots where T not wanted • Light shines on mask • T added • Continue for all four bases • Need 80 masks for total 20-mer oligonucleotide light Ink-jet printer microarrays – Ink-jet printhead draws up DNA – Printhead moves to specific location on solid support – DNA ejected through small hole – Used to spot DNA or synthesize oligonucleotides directly on glass slide – Use pioneered by Agilent Technologies, Inc. Comparisons of microarrays Comparison of microarray hybridization • Spotted microarrays – Competitive hybridization • Two labeled cDNAs hybridized to same slide • Affymetrix GeneChips – One labeled RNA population per chip – Comparison made between hybridization intensities of same oligonucleotides on different chips Target labeling: fluorescent cDNA • cDNA made using reverse transcriptase • Fluorescently labeled nucleotides added • Labeled nucleotides incorporated into cDNA Target labeling: cRNA + biotin • cDNA made with reverse transcriptase • Linker added with T7 RNA polymerase recognition site • T7 polymerase added and biotin labeled RNA bases • Biotin label incorporated into cRNA + Labels • Cy3 and Cy5 – Fluoresce at different wavelengths – Used for competitive hybridization • Biotin – Binds to fluorescently labeled avidin – Used with Affymetrix GeneChips Spotted-microarray hybridization • Control and experimental cDNA labeled – One sample labeled with Cy3 – Other sample labeled with Cy5 • Both samples hybridized together to microarray • Relative intensity determined using confocal laser scanner Scanning of microarrays laser • Confocal laser scanning microscopy • Laser beam excites each spot of DNA • Amount of fluorescence detected • Different lasers used for different wavelengths – Cy3 – Cy5 detection Analysis of hybridization • Results given as ratios • Images use colors: Cy3 = Green Cy5 = red Yellow – Yellow is equal intensity or no change in expression Example of spotted microarray • RNA from irradiated cells (red) • Compare with untreated cells (green) • Most genes have little change (yellow) • Gene CDKN1A: red = increase in expression • Gene Myc: green = decrease in expression CDKNIA MYC -Flash animation -YouTube video Analysis of cell-cycle regulation • Yeast cells stopped at different stages of cell cycle – G1, S, G2, and M • RNA extracted from each stage • Control RNA from unsynchronized culture Results of yeast cell-cycle analysis • 800 genes identified whose expression changes during cell cycle • Grouped by peak expression • M/G1, G1, S, G2, and M • Four different treatments used to synchronize cells – All gave similar results • Results from Spellman et al., 1998; Cho et al., 1998 Cell-cycle regulated genes Alpha • Each gene is a line on the longitudinal axis • Treatments in different panels • Cell-cycle stages are color coded at top • Vertical axis groups genes by stage in which expression peaks cdc15 cdc28 Elu M/G1 G1 S G2 M Brown and Botstein, 1999 Affymetrix GeneChip experiment • RNA from different types of brain tumors extracted • Extracted RNA hybridized to GeneChips containing approximately 6,800 human genes • Identified gene expression profiles specific to each type of tumor Profiling tumors • Image portrays gene expression profiles showing differences between different tumors • Tumors: MD (medulloblastoma) Mglio (malignant glioma) Rhab (rhabdoid) PNET (primitive neuroectodermal tumor) • Ncer: normal cerebella • Gene expression differences for medulloblastoma correlated with response to chemotherapy • Those who failed to respond had a different profile from survivors • Can use this approach to determine treatment 60 different samples Cancer diagnosis by microarray Analysis of microarray results • Inherent variability: need for repetition – Biological and technical replicates • Analysis algorithms – Based on statistical models • Means of generating hypotheses that need to be tested