© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Chapter 5 RNA Expression Analysis Determining genomewide RNA expression levels

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 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 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 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 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 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

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 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 Northern blots vs. microarrays  Global expression analysis: microarrays  RNA levels of every gene in the genome analyzed in parallel  Global expression analysis: Northern blot  Limited by number of lanes in gel target – loading – control

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 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 Microarray hybridization  Usually comparative  Ratio between two samples  Examples  Tumor vs. normal tissue  Drug treatment vs. no treatment  Embryo vs. adult mRNA cDNA DNA microarray samples

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 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 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 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 Commercial DNA spotter

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Movie of microarray spotting

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 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 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 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 lampmaskchip

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 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 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 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 Comparisons of microarrays

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 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 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 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 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 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 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

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 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 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 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 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 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 Brown and Botstein, 1999 Alphacdc15cdc28Elu M/G1 G1 S G2 M

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 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 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 Cancer diagnosis by microarray  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

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 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 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

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Schematic of SAGE method:

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey SAGE II  Cleave cDNAs with four-base cutter restriction enzyme  Ligate adapters containing site for type- IIs restriction enzyme  Cut 14 base pairs from recognition site CATG GTAC TTTTTTT CATG GTAC TTTTTTT GTAC AAAAAAA TTTTTTT AAAAAAA TTTTTTT AAAAAAA

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 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 CATG GTAC GGTCAC CCAGTG CATG GTAC GGTCAC CCAGTG

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 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 MPSS I  Massively parallel signature sequencing  Means of determining abundance of RNA species  Unique tags added to cDNAs  Tags hybridized to oligonucleotides on microbeads

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey MPSS II  Sequencing performed in glass chamber  Initiated by restriction enzyme revealing four- base 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 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 Real-time PCR readout  The readout of a real- time 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 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 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