1 / 48

Chapter 5 RNA Expression Analysis

Chapter 5 RNA Expression Analysis. Determining genome-wide RNA expression levels. Contents. Genome-wide RNA expression analysis Northern blotting Types of microarrays Making microarrays Hybridization to microarrays Microarray experiments SAGE (Serial Analysis of Gene Expression),

oakley
Télécharger la présentation

Chapter 5 RNA Expression Analysis

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Chapter 5RNA Expression Analysis Determining genome-wide RNA expression levels © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  2. Contents • Genome-wide RNA expression analysis • Northern blotting • Types of microarrays • Making microarrays • Hybridization to microarrays • Microarray experiments • SAGE (Serial Analysis of Gene Expression), • MPSS™ (Massively Parallel Signature Sequencing), • Real-time RT-PCR © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  3. Genome-wide expression analysis • Goal: to measure RNA levels of all genes in genome • Steady-state RNA levels vary with the following: • Cell type • Developmental stage: program • External stimuli: heat and cold etc. • Time (when) and location (where) of expression provide useful information as to gene function. Temporal and spatial • Misconception: More is Merrier! • Problem: Its only RNA not protein © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  4. 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

  5. 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

  6. 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 07458

  7. 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

  8. target – loading – control 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 © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  9. 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

  10. samples mRNA cDNA DNA microarray Microarray hybridization • Usually comparative • Ratio between two samples • Examples • Tumor vs. normal tissue • Drug treatment vs. no treatment • Embryo vs. adult © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  11. 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

  12. 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

  13. 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/DNA 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

  14. Commercial DNA spotter © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  15. Movie of microarray spotting © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  16. 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

  17. 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 • Non-overlapping • Composition based on design rules • Empirically derived © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  18. lamp mask chip 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 © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  19. light 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 © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  20. light 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 © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  21. 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

  22. Comparisons of microarrays Oligo spotted Ink jet © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  23. Comparison of microarray hybridization • Spotted microarrays • Competitive hybridization • Two labeled cDNAs hybridized to the 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

  24. 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

  25. 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

  26. 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

  27. 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 • Reverse the label © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  28. 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 07458

  29. 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

  30. 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

  31. 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

  32. 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

  33. Alpha cdc15 cdc28 Elu M/G1 G1 S G2 M Brown and Botstein, 1999 Cell-cycle regulated genes treatments • 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 © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  34. 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

  35. 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

  36. 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 07458

  37. 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 • real-time PCR • Reverse genetics by knockouts © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  38. SAGE Ihttp://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

  39. AAAAAAA TTTTTTT AAAAAAA TTTTTTT GTAC TTTTTTT CATG GTAC AAAAAAA TTTTTTT CATG GTAC AAAAAAA 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 © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  40. CATG GTAC GGTCAC CCAGTG CATG GTAC GGTCAC CCAGTG CATG GTAC GGTCAC CCAGTG CATG GTAC 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 © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  41. 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

  42. Schematic of SAGE method: © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  43. MPSS I (Lynx Therapeutics, Inc) • 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

  44. 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 (costs 25K) Up to 1,000,000 sequencing reactions are done at once © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

  45. 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

  46. 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 07458

  47. 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 correlation 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

  48. 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

More Related