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Chapter 5 RNA Expression Analysis Determining genomewide RNA expression levels © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Contents Genomewide RNA expression analysis Northern blotting Types of microarrays Making microarrays Hybridization to microarrays Microarray experiments SAGE MPSS Real-time PCR © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 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 Misconception: More is Merrier! © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Genomics expression analysis methods Microarrays Hybridization based SAGE (Serial Analysis of Gene Expression) Sequence fragments of cDNAs MPSS (Massively Parallel Signature Sequencing) Combines hybridization and sequencing Real-time PCR © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 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) © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Northern blot – Electrophoresis of RNA through gel + gel Transfer of RNA to solid support Nylon or nitrocellulose Intensity of hybridization signal Approximately equal to amount of RNA © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Hybridization issues RNA integrity must be verified If RNA degraded, hybridization not quantitative Probe must be in excess of bound RNA Hybridization kinetics govern reaction Hybridization must be for a sufficient time to allow probe to find target RNA Comparison between samples requires loading control © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Northern blots vs. microarrays Global expression analysis: microarrays RNA levels of every gene in the genome analyzed in parallel target – loading – control Global expression analysis: Northern blot Limited by number of lanes in gel © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 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” © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Microarray hybridization Usually comparative Ratio between two samples samples mRNA Examples Tumor vs. normal tissue Drug treatment vs. no treatment Embryo vs. adult cDNA DNA microarray © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 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 © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 DNA spotting I DNA spotting usually uses multiple pins DNA in microtiter plate DNA usually PCR amplified Oligonucleotides can also be spotted © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 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 © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Commercial DNA spotter © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Movie of microarray spotting QuickTime™ and a YUV420 codec decompressor are needed to see this picture. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 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 © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 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 © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Photolithography Light-activated chemical reaction For addition of bases to growing oligonucleotide Custom masks Prevent light from reaching spots where bases not wanted lamp mask chip Mirrors also used NimbleGen™ uses this approach © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Example: building oligonucleotides by photolithography 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 light © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 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 © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 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. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Comparisons of microarrays © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 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 © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Target labeling: fluorescent cDNA cDNA made using reverse transcriptase Fluorescently labeled nucleotides added Labeled nucleotides incorporated into cDNA © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 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 + © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Labels Cy3 green and Cy5 red Fluoresce at different wavelengths Used for competitive hybridization Biotin Binds to fluorescently labeled avidin Used with Affymetrix GeneChips © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 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 © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Scanning of microarrays Confocal laser scanning microscopy Laser beam excites each spot of DNA Amount of fluorescence detected Different lasers used for different wavelengths laser detection Cy3 Cy5 © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Analysis of hybridization Results given as ratios Images use colors: Cy3 = Green Cy5 = red Yellow Yellow is equal intensity or no change in expression © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 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 © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 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 © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Results of cell-cycle analysis 800/6000 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 © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Cell-cycle regulated genes 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 Alpha cdc15 cdc28 Elu M/G1 G1 S G2 M Brown and Botstein, 1999 © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 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 © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 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 © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 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 © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 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 © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 SAGE I http://www.sagenet.org/ 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 © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Schematic of SAGE method: © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 SAGE II Cleave cDNAs with four-base cutter restriction enzyme Ligate adapters containing site for typeIIs restriction enzyme Cut 14 base pairs from recognition site AAAAAAA TTTTTTT GTAC AAAAAAA TTTTTTT CATG GTAC AAAAAAA TTTTTTT CATG GTAC AAAAAAA TTTTTTT © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 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 © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 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 © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 MPSS I Massively parallel signature sequencing Means of determining abundance of RNA species Unique tags added to cDNAs Tags hybridized to oligonucleotides on microbeads http://www.lynxgen.com/wt/tert.php3?page_name=mpss © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 MPSS II Sequencing performed in glass chamber Initiated by restriction enzyme revealing fourbase overhang Hybridization of four-base adapters used to read sequence Number of times a particular sequence is found is measure of RNA abundance © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 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 © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 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 © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 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 © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Summary Microarrays Compared with Northern blots How they are made How they are used Differences between spotted and oligonucleotide microarrays Examples of microarray experiments SAGE MPSS Real-time PCR © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458