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Genomics I: The Transcriptome RNA Expression Analysis Determining genomewide RNA expression levels Contents Genomewide RNA expression analysis Northern blotting Macroarrays Microarrays Making microarrays Hybridization to microarrays Microarray experiments SAGE Real-time PCR 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 SAGE (Serial Analysis of Gene Expression) Sequence fragments of cDNAs MPSS (Massively Parallel Signature Sequencing) Hybridization based Combines hybridization and sequencing Real-time PCR Hybridization Measurements of RNA abundance by microarrays based on hybridization Between complementary strands of RNA and DNA Or two complementary DNA strands Similar in principle to RNA blot (Northern blot) Northern blot – Electrophoresis of RNA through gel Transfer of RNA to solid support Nylon or nitrocellulose Intensity of hybridization signal Approximately equal to amount of RNA gel + Hybridization issues RNA integrity must be verified Probe must be in excess of bound RNA Hybridization kinetics govern reaction If RNA degraded, hybridization not quantitative Hybridization must be for a sufficient time to allow probe to find target RNA Comparison between samples requires loading control Macroarray Analysis Macroarray Analysis Microarray Analysis of Transcription Animation Northern blots vs. microarrays 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 target – loading – control Basics of microarrays DNA attached to solid support RNA is labeled Glass, plastic, or nylon 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 Same spots usually printed at different locations DNA sticks to slide by hydrostatic interactions 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 Oligonucleotides for each gene selected by computer program to be the following: Usually 20–25 bases in length 10–20 different oligonucleotides for each gene Unique in genome Nonoverlapping Composition based on design rules Empirically derived Photolithography Light-activated chemical reaction Custom masks For addition of bases to growing oligonucleotide 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 Confocal laser scanning microscopy Laser beam excites each spot of DNA Amount of fluorescence detected Different lasers used for different wavelengths Cy3 Cy5 laser 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 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 Four different treatments used to synchronize cells M/G1, G1, S, G2, and M 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 Analysis algorithms Biological and technical replicates Based on statistical models Means of generating hypotheses that need to be tested SAGE I Serial analysis of gene expression Concept: sequence a small piece of each cDNA in a library Gives measure of abundance of each RNA species Method Cut off “tag” from each cDNA Ligate tags together into a concatemer Sequence the concatemer SAGE II Cleave cDNAs with fourbase cutter restriction enzyme AAAAAAA TTTTTTT GTAC AAAAAAA TTTTTTT Ligate adapters containing site for typeIIs restriction enzyme CATG GTAC AAAAAAA TTTTTTT Cut 14 base pairs from recognition site CATG GTAC AAAAAAA TTTTTTT SAGE III Ligate on adapters with restriction sites Cut with two restriction enzymes to release 26 base pair tag Ligate tags together into ~500 base pair concatemer CATG GTAC GGTCAC CCAGTG CATG GTAC GGTCAC CCAGTG CATG GTAC GGTCAC CCAGTG CATG GTAC SAGE IV Sequence the concatemers Identify tag borders Size of tag and restriction-enzyme sites Compare tag sequences to database Abundance of tag is measure of abundance of that RNA species Real-time PCR Sensitive means of measuring RNA abundance Not genomewide: used to verify microarray results TaqMan method uses fluorescently tagged primers Fluorescent tag released by Taq polymerase Real-time PCR readout The readout of a realtime PCR reaction is a set of curves The curves indicate the PCR cycle at which fluorescence is detected Each cycle is twice the amount of the previous cycle Genomic analysis of gene expression Methods capable of giving a “snapshot” of RNA expression of all genes Can be used as diagnostic profile Example: cancer diagnosis Can show how RNA levels change during development, after exposure to stimulus, during cell cycle, etc. Provides large amounts of data Can help us start to understand how whole systems function