1. Genomics I: The Transcriptome RNA Expression Analysis Determining genomewide RNA expression levels

2. Contents Genomewide RNA expression analysis Genomewide RNA expression analysis Northern blotting Northern blotting Macroarrays Macroarrays Microarrays Microarrays Making microarrays Making microarrays Hybridization to microarrays Hybridization to microarrays Microarray experiments Microarray experiments SAGE SAGE Real-time PCR Real-time PCR

3. Genomewide expression analysis Goal: to measure RNA levels of all genes in genome Goal: to measure RNA levels of all genes in genome RNA levels vary with the following: RNA levels vary with the following: Cell type Cell type Developmental stage Developmental stage External stimuli External stimuli Time and location of expression provide useful information as to gene function Time and location of expression provide useful information as to gene function

4. Genomics expression analysis methods Microarrays Microarrays Hybridization based Hybridization based SAGE (Serial Analysis of Gene Expression) SAGE (Serial Analysis of Gene Expression) Sequence fragments of cDNAs Sequence fragments of cDNAs MPSS (Massively Parallel Signature Sequencing) MPSS (Massively Parallel Signature Sequencing) Combines hybridization and sequencing Combines hybridization and sequencing Real-time PCR Real-time PCR

5. Hybridization Measurements of RNA abundance by microarrays based on hybridization Measurements of RNA abundance by microarrays based on hybridization Between complementary strands of RNA and DNA Between complementary strands of RNA and DNA Or two complementary DNA strands Or two complementary DNA strands Similar in principle to RNA blot (Northern blot) Similar in principle to RNA blot (Northern blot)

6. Northern blot Electrophoresis of RNA through gel Electrophoresis of RNA through gel Transfer of RNA to solid support Transfer of RNA to solid support Nylon or nitrocellulose Nylon or nitrocellulose Intensity of hybridization signal Intensity of hybridization signal Approximately equal to amount of RNA Approximately equal to amount of RNA – + gel

7. Hybridization issues RNA integrity must be verified RNA integrity must be verified If RNA degraded, hybridization not quantitative If RNA degraded, hybridization not quantitative Probe must be in excess of bound RNA Probe must be in excess of bound RNA Hybridization kinetics govern reaction Hybridization kinetics govern reaction Hybridization must be for a sufficient time to allow probe to find target RNA Hybridization must be for a sufficient time to allow probe to find target RNA Comparison between samples requires loading control Comparison between samples requires loading control

8. Macroarray Analysis

10. Microarray Analysis of Transcription

11. Animation

12. Northern blots vs. microarrays Global expression analysis: Northern blot Global expression analysis: Northern blot Limited by number of probes that can be used simultaneously Limited by number of probes that can be used simultaneously Global expression analysis: microarrays Global expression analysis: microarrays RNA levels of every gene in the genome analyzed in parallel RNA levels of every gene in the genome analyzed in parallel target – loading – control

13. Basics of microarrays DNA attached to solid support DNA attached to solid support Glass, plastic, or nylon Glass, plastic, or nylon RNA is labeled RNA is labeled Usually indirectly Usually indirectly Bound DNA is the probe Bound DNA is the probe Labeled RNA is the “target” Labeled RNA is the “target”

14. Microarray hybridization Usually comparative Usually comparative Ratio between two samples Ratio between two samples Examples Examples Tumor vs. normal tissue Tumor vs. normal tissue Drug treatment vs. no treatment Drug treatment vs. no treatment Embryo vs. adult Embryo vs. adult mRNA cDNA DNA microarray samples

15. Two major types of microarrays cDNA arrays- PCR product corresponding to a portion of a cDNA is immobilized on the slide 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 oligonucleotide arrays- oligonucleotide complementary to transcript is synthesized on slide or immobilized on the slide

16. How microarrays are made: spotted microarrays DNA mechanically placed on glass slide DNA mechanically placed on glass slide Need to deliver nanoliter to picoliter volumes Need to deliver nanoliter to picoliter volumes Too small for normal pipetting devices Too small for normal pipetting devices Robot “prints,” or “spots,” DNA in specific places Robot “prints,” or “spots,” DNA in specific places

17. DNA spotting I DNA spotting usually uses multiple pins DNA spotting usually uses multiple pins DNA in microtiter plate DNA in microtiter plate DNA usually PCR amplified DNA usually PCR amplified Oligonucleotides can also be spotted Oligonucleotides can also be spotted

18. DNA spotting II Pins dip into DNA solution in microtiter wells Pins dip into DNA solution in microtiter wells Robot moves pins with DNA to slides Robot moves pins with DNA to slides Robot “prints” DNA onto slide Robot “prints” DNA onto slide DNA sticks to slide by hydrostatic interactions DNA sticks to slide by hydrostatic interactions Same spots usually printed at different locations Same spots usually printed at different locations Serves as internal control Serves as internal control Pins washed between printing rounds Pins washed between printing rounds Hundreds of slides can be printed in a day Hundreds of slides can be printed in a day

19. Commercial DNA spotter

20. How microarrays are made: Affymetrix GeneChips Oligonucleotides synthesized on silicon chip Oligonucleotides synthesized on silicon chip One base at a time One base at a time Uses process of photolithography Uses process of photolithography Developed for printing computer circuits Developed for printing computer circuits

21. Affymetrix GeneChips Oligonucleotides Oligonucleotides Usually 20–25 bases in length Usually 20–25 bases in length 10–20 different oligonucleotides for each gene 10–20 different oligonucleotides for each gene Oligonucleotides for each gene selected by computer program to be the following: Oligonucleotides for each gene selected by computer program to be the following: Unique in genome Unique in genome Nonoverlapping Nonoverlapping Composition based on design rules Composition based on design rules Empirically derived Empirically derived

22. Photolithography Light-activated chemical reaction Light-activated chemical reaction For addition of bases to growing oligonucleotide For addition of bases to growing oligonucleotide Custom masks Custom masks Prevent light from reaching spots where bases not wanted Prevent light from reaching spots where bases not wanted Mirrors also used Mirrors also used NimbleGen™ uses this approach NimbleGen™ uses this approach lampmaskchip

23. Example: building oligonucleotides by photolithography Want to add nucleotide G Want to add nucleotide G Mask all other spots on chip Mask all other spots on chip Light shines only where addition of G is desired Light shines only where addition of G is desired G added and reacts G added and reacts Now G is on subset of oligonucleotides Now G is on subset of oligonucleotides light

24. Example: adding a second base Want to add T Want to add T New mask covers spots where T not wanted New mask covers spots where T not wanted Light shines on mask Light shines on mask T added T added Continue for all four bases Continue for all four bases Need 80 masks for total Need 80 masks for total 20-mer oligonucleotide light

25. Ink-jet printer microarrays Ink-jet printhead draws up DNA Ink-jet printhead draws up DNA Printhead moves to specific location on solid support Printhead moves to specific location on solid support DNA ejected through small hole DNA ejected through small hole Used to spot DNA or synthesize oligonucleotides directly on glass slide Used to spot DNA or synthesize oligonucleotides directly on glass slide Use pioneered by Agilent Technologies, Inc. Use pioneered by Agilent Technologies, Inc.

26. Comparisons of microarrays

27. Comparison of microarray hybridization Spotted microarrays Spotted microarrays Competitive hybridization Competitive hybridization Two labeled cDNAs hybridized to same slide Two labeled cDNAs hybridized to same slide Affymetrix GeneChips Affymetrix GeneChips One labeled RNA population per chip One labeled RNA population per chip Comparison made between hybridization intensities of same oligonucleotides on different chips Comparison made between hybridization intensities of same oligonucleotides on different chips

28. Target labeling: fluorescent cDNA cDNA made using reverse transcriptase cDNA made using reverse transcriptase Fluorescently labeled nucleotides added Fluorescently labeled nucleotides added Labeled nucleotides incorporated into cDNA Labeled nucleotides incorporated into cDNA

29. Target labeling: cRNA + biotin cDNA made with reverse transcriptase cDNA made with reverse transcriptase Linker added with T7 RNA polymerase recognition site Linker added with T7 RNA polymerase recognition site T7 polymerase added and biotin labeled RNA bases T7 polymerase added and biotin labeled RNA bases Biotin label incorporated into cRNA Biotin label incorporated into cRNA +

30. Labels Cy3 and Cy5 Cy3 and Cy5 Fluoresce at different wavelengths Fluoresce at different wavelengths Used for competitive hybridization Used for competitive hybridization Biotin Biotin Binds to fluorescently labeled avidin Binds to fluorescently labeled avidin Used with Affymetrix GeneChips Used with Affymetrix GeneChips

31. Spotted-microarray hybridization Control and experimental cDNA labeled Control and experimental cDNA labeled One sample labeled with Cy3 One sample labeled with Cy3 Other sample labeled with Cy5 Other sample labeled with Cy5 Both samples hybridized together to microarray Both samples hybridized together to microarray Relative intensity determined using confocal laser scanner Relative intensity determined using confocal laser scanner

32. Scanning of microarrays Confocal laser scanning microscopy Confocal laser scanning microscopy Laser beam excites each spot of DNA Laser beam excites each spot of DNA Amount of fluorescence detected Amount of fluorescence detected Different lasers used for different wavelengths Different lasers used for different wavelengths Cy3 Cy3 Cy5 Cy5 laser detection

33. Results given as ratios Results given as ratios Images use colors: Images use colors: Cy3 = Green Cy5 = red Yellow Yellow is equal intensity or no change in expression Yellow is equal intensity or no change in expression Analysis of hybridization

34. Example of spotted microarray RNA from irradiated cells (red) RNA from irradiated cells (red) Compare with untreated cells (green) Compare with untreated cells (green) Most genes have little change (yellow) Most genes have little change (yellow) Gene CDKN1A: red = increase in expression Gene CDKN1A: red = increase in expression Gene Myc: green = decrease in expression Gene Myc: green = decrease in expression CDKNIA MYC

35. Analysis of cell-cycle regulation Yeast cells stopped at different stages of cell cycle Yeast cells stopped at different stages of cell cycle G1, S, G2, and M G1, S, G2, and M RNA extracted from each stage RNA extracted from each stage Control RNA from unsynchronized culture Control RNA from unsynchronized culture

36. Results of yeast cell-cycle analysis 800 genes identified whose expression changes during cell cycle 800 genes identified whose expression changes during cell cycle Grouped by peak expression Grouped by peak expression M/G1, G1, S, G2, and M M/G1, G1, S, G2, and M Four different treatments used to synchronize cells Four different treatments used to synchronize cells All gave similar results All gave similar results Results from Spellman et al., 1998; Cho et al., 1998 Results from Spellman et al., 1998; Cho et al., 1998

37. Cell-cycle regulated genes Each gene is a line on the longitudinal axis Each gene is a line on the longitudinal axis Treatments in different panels Treatments in different panels Cell-cycle stages are color coded at top Cell-cycle stages are color coded at top Vertical axis groups genes by stage in which expression peaks Vertical axis groups genes by stage in which expression peaks Brown and Botstein, 1999 Alphacdc15cdc28Elu M/G1 G1 S G2 M

38. Affymetrix GeneChip experiment RNA from different types of brain tumors extracted RNA from different types of brain tumors extracted Extracted RNA hybridized to GeneChips containing approximately 6,800 human genes Extracted RNA hybridized to GeneChips containing approximately 6,800 human genes Identified gene expression profiles specific to each type of tumor Identified gene expression profiles specific to each type of tumor

39. Profiling tumors Image portrays gene expression profiles showing differences between different tumors Image portrays gene expression profiles showing differences between different tumors Tumors: Tumors: MD (medulloblastoma) Mglio (malignant glioma) Rhab (rhabdoid) PNET (primitive neuroectodermal tumor) Ncer: normal cerebella Ncer: normal cerebella

40. Cancer diagnosis by microarray Gene expression differences for medulloblastoma correlated with response to chemotherapy Gene expression differences for medulloblastoma correlated with response to chemotherapy Those who failed to respond had a different profile from survivors Those who failed to respond had a different profile from survivors Can use this approach to determine treatment Can use this approach to determine treatment 60 different samples

41. Analysis of microarray results Inherent variability: need for repetition Inherent variability: need for repetition Biological and technical replicates Biological and technical replicates Analysis algorithms Analysis algorithms Based on statistical models Based on statistical models Means of generating hypotheses that need to be tested Means of generating hypotheses that need to be tested

42. SAGE I Serial analysis of gene expression Serial analysis of gene expression Concept: sequence a small piece of each cDNA in a library Concept: sequence a small piece of each cDNA in a library Gives measure of abundance of each RNA species Gives measure of abundance of each RNA species Method Method Cut off “tag” from each cDNA Cut off “tag” from each cDNA Ligate tags together into a concatemer Ligate tags together into a concatemer Sequence the concatemer Sequence the concatemer

43. SAGE II Cleave cDNAs with four- base cutter restriction enzyme Cleave cDNAs with four- base cutter restriction enzyme Ligate adapters containing site for type- IIs restriction enzyme Ligate adapters containing site for type- IIs restriction enzyme Cut 14 base pairs from recognition site Cut 14 base pairs from recognition site CATG GTAC TTTTTTT CATG GTAC TTTTTTT GTAC AAAAAAA TTTTTTT AAAAAAA TTTTTTT AAAAAAA

44. SAGE III Ligate on adapters with restriction sites Ligate on adapters with restriction sites Cut with two restriction enzymes to release 26 base pair tag Cut with two restriction enzymes to release 26 base pair tag Ligate tags together into ~500 base pair concatemer Ligate tags together into ~500 base pair concatemer CATG GTAC GGTCAC CCAGTG CATG GTAC CATG GTAC GGTCAC CCAGTG CATG GTAC GGTCAC CCAGTG

45. SAGE IV Sequence the concatemers Sequence the concatemers Identify tag borders Identify tag borders Size of tag and restriction-enzyme sites Size of tag and restriction-enzyme sites Compare tag sequences to database Compare tag sequences to database Abundance of tag is measure of abundance of that RNA species Abundance of tag is measure of abundance of that RNA species

46. Real-time PCR Sensitive means of measuring RNA abundance Sensitive means of measuring RNA abundance Not genomewide: used to verify microarray results Not genomewide: used to verify microarray results TaqMan method uses fluorescently tagged primers TaqMan method uses fluorescently tagged primers Fluorescent tag released by Taq polymerase Fluorescent tag released by Taq polymerase

47. Real-time PCR readout The readout of a real- time PCR reaction is a set of curves The readout of a real- time PCR reaction is a set of curves The curves indicate the PCR cycle at which fluorescence is detected The curves indicate the PCR cycle at which fluorescence is detected Each cycle is twice the amount of the previous cycle Each cycle is twice the amount of the previous cycle

48. Genomic analysis of gene expression Methods capable of giving a “snapshot” of RNA expression of all genes Methods capable of giving a “snapshot” of RNA expression of all genes Can be used as diagnostic profile Can be used as diagnostic profile Example: cancer diagnosis Example: cancer diagnosis Can show how RNA levels change during development, after exposure to stimulus, during cell cycle, etc. Can show how RNA levels change during development, after exposure to stimulus, during cell cycle, etc. Provides large amounts of data Provides large amounts of data Can help us start to understand how whole systems function Can help us start to understand how whole systems function