Genomics I: The Transcriptome

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

2. 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

3. Genomics expression analysis methods • Microarrays • Hybridization based • RNA-seq • Direct sequencing of cDNAs • SAGE (Serial Analysis of Gene Expression) • Sequence fragments of cDNAs • Real-time PCR

4. Macroarray Analysis

5. Macroarray Analysis

6. Microarray Analysis of Transcription

7. Animation

8. target – loading – control 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

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”

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

11. 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

12. 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

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

14. 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

15. Commercial DNA spotter

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

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 • Nonoverlapping • Composition based on design rules • Empirically derived

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

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

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

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.

22. Comparisons of microarrays

23. 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

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

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 +

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

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

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

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

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 -Flash animation -YouTube video

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

32. Results of yeast cell-cycle analysis • 800 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

33. Alpha cdc15 cdc28 Elu M/G1 G1 S G2 M Brown and Botstein, 1999 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

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

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

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

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