Microarrays, RNAseq And Functional Genomics CPSC265 Matt Hudson

1. Microarrays, RNAseq AndFunctional Genomics CPSC265 Matt Hudson

2. Microarray Technology • Relatively young technology – • Already mostly obsolete, though. • Usually used like a Northern blot – can determine the amount of mRNA for a particular gene • Except – a Northern blot measures one gene at a time • A microarray can measure every gene in the genome, simultaneously

3. Recent! History • 1994. First microarrays developed by Ron Davis and Pat Brown at Stanford. • 1997-1999. Practical microarrays become available for yeast, humans and plants

4. Why analyze so many genes? • Just because we sequenced a genome doesn’t mean we know anything about the genes. Thousands of genes remain without an assigned function. • To find genes involved in a particular process, we can look for mRNAs “up-regulated” during that process. • For example, we can look at genes up-regulated in human cells in response to cancer-causing mutations, or look at genes in a crop plant responding to drought. • Patterns/clusters of expression are more predictive than looking at one or two prognostic markers – can figure out new pathways

5. Two Main Types of Microarray • Oligonucleotide, photolithographic arrays • “Gene Chips” • Miniaturized, high density arrays of oligos • (Affymetrix Inc., Nimblegen, Inc.) • Printed cDNA or Oligonucleotide Arrays • Robotically spotted cDNAs or Oligonucleotides • Printed on Nylon, Plastic or Glass surface • Can be made in any lab with a robot • Several robots in ERML • Can also buy printed arrays commercially

6. The original ideaA microarray of thousands of genes on a glass slideEach “spot” is one gene, like a probe in a Northern blot.This time, the probes are fixed, and the target genes move about.

7. Glass slide microarray summary

8. Theprocess Building the chip: PCR PURIFICATION and PREPARATION MASSIVE PCR PREPARING SLIDES PRINTING RNA preparation: Hybing the chip: CELL CULTURE AND HARVEST POST PROCESSING ARRAY HYBRIDIZATION RNA ISOLATION DATA ANALYSIS PROBE LABELING cDNA PRODUCTION

9. Robotically printed arrays chemically modified slides 384 well source plate steel spotting pin 1 nanolitre spots 90-120 um diameter

10. Physical Spotting

12. Labelling RNA for Glass slides Reverse Transcriptase Reverse transcription cDNA Cy3 labelled mRNA (control) Cy3 label mRNA (treated) cDNA Cy5 labelled Cy5 label

13. HybridizationBinding of cDNA target samples to cDNA probes on the slide cover slip Hybridize for 5-12 hours

14. Northern blot vs. Microarray • In Northern blotting, the whole mRNA of the organism is on the membrane. The labelled “probe” lights up a band – one gene • In a microarray, the whole genome is printed on a slide, one “probe” spot per gene. Mixed, labelled cDNA, made from mRNA from the organism, is added. Each probe lights up green or red according to whether it is more or less abundant between the control and the treated mRNA.

15. Hybridization chamber 3XSSC • Humidity • Temperature • Formamide (Lowers the Tm) HYB CHAMBER ARRAY LIFTERSLIP SLIDE LABEL SLIDE LABEL

16. Expression profiling with DNA microarrays cDNA “B” Cy3 labeled cDNA “A” Cy5 labeled Laser 1 Laser 2 Scanning Hybridization + Analysis Image Capture

17. Image analysis GenePix

18. Spotted cDNA microarrays Advantages • Lower price and flexibility • Can be printed in well equipped lab • Simultaneous comparison of two related biological samples (tumor versus normal, treated versus untreated cells) Disadvantages • Needs sequence verification • Measures the relative level of expression between 2 samples

19. Affymetrix Microarrays • One chip per sample • Made by photolithography • ~500,000 25 base probes • …unlike Glass Slide Microarrays • Made by a spotting robot • ~30,000 50-500 base probes • Involves two dyes/one chip • Control and experiment • on same chip

20. Affymetrix GeneChip • Miniaturized, high density arrays of oligos • 1.28-cm by 1.28-cm (409,000 oligos) • Manufacturing Process • Solid-phase chemical synthesis and Photolithographic fabrication techniques employed in semiconductor industry

21. Selection of Expression ProbesSet of oligos to be synthesized is defined, based on its ability to hybridize to the target genes of interest 3’ 5’ Sequence Probes Perfect Match Mismatch Chip Computer algorithms are used to design photolithographic masks for use in manufacturing

23. Photolithographic Synthesis Manufacturing ProcessProbe arrays are manufactured by light-directed chemical synthesis process which enables the synthesis of hundreds of thousands of discrete compounds in precise locations Lamp Mask Chip

24. Affymetrix Wafer and Chip Format 20 - 50 µm 50… 11µm Millions of identical oligonucleotides per feature 49 - 400 chips/wafer 1.28cm up to ~ 400,000 “features” / chip

25. Labelling RNA for Affymetrix Reverse Transcriptase Reverse transcription cDNA mRNA in vitro transcription Transcription Biotin labelled nucleotides cRNA

26. Biotin-labeled transcripts B B B B B B B B Fragmented cRNA Target Preparation Fragment (heat, Mg2+) cDNA Wash & Stain Scan AAAA mRNA Hybridize (16 hours)

27. Hybridized Array cRNA Target Streptravidin-phycoerythrin conjugate GeneChip® Expression AnalysisHybridizationand Staining Array

28. Example: Comparing a mutant cellline with awild typeline.

29. Instrumentation Affymetrix GeneChip System 3000-7G Scanner 450 Fluidic Station

30. Microarray data analysis This is now a very important branch of statistics It is unusual to do thousands of experiments at once. Statistical methods didn’t exist to analyse microarrays. Now they are being rapidly developed.

31. Normal vs. Normal Normal vs. Tumor

32. Lung Tumor: Up-Regulated Lung Tumor: Down-Regulated

33. Microarray Technology - Applications • Gene Discovery- • Assigning function to sequence • Finding genes involved in a particular process • Discovery of disease genes and drug targets • Genotyping • SNPs • Genetic mapping (Humans, plants) • Patient stratification (pharmacogenomics) • Adverse drug effects (ADE) • Microbial ID

34. Why it is becoming obsolete • In a word, RNAseq • RNAseq uses DNA sequencing to do the same thing. • Rather than an array, you just sequence millions of mRNA fragments, then figure out what genes they are from

35. Why RNAseq only just caught on • It’s been around for a long time, called things like SAGE and MPSS. • But they were expensive and arrays were cheap. Now, sequencing is as cheap as arrays • Also, you need a fully sequenced reference genome for the computer analysis.

36. What RNAseq / arrays can’t do • Tell you anything about protein levels • Tell you anything about post-translational modification of proteins • Tell you anything about the structure of proteins • Predict the phenotype of a genetic mutant

37. Proteomics • A high througput approach to learning about all the proteins in a cell • As microarrays are to a Northern blot, proteomics is to a Western blot • Two main approaches – • 2D gels + MS • Protein microarrays

38. pI pH 3 pH 10 kDa Protein separation: 2-dimensional gel electrophoresis 1st dimension Separation by charge (isoelectric focussing) 2nd dimension Separation by molecular weight (SDS-PAGE) Susan Liddel

39. Proteins extracted from cow ovarian follicle granulosa cells separated on a broad range IPG strip (pH3-10) followed by a 12.5% polyacrylamide gel, silver stained 3.5 9.0 150 100 75 50 37 25 20 Susan Liddel

40. Mass Spectrometry FT-MS can tell you10-20 residues ofsequence, but onlyfrom a purified protein Robots pick spots from2-D gel, load into MS Also, 2-D and 3-D LC

41. Array-based protein interaction detection

42. Protein microarrays

43. The future of microarrays: • Still looking good, in areas other than research • Used by pharmaceutical companies, medical diagnostics, etc. • In the future, just like silicon chips, likely • to get cheaper, faster and more powerful • It may not be long before they are routinely used to diagnose disease

44. The future of proteomics: • Many people will tell you proteomics IS the future of biology • If they can get it to work as well as microarrays, they will be right • The problem is, every protein has different chemistry, while all mRNAs are closely comparable • At the moment, proteomics is a hot field, but few major biological discoveries have been made with proteomics – many have been made with microarrays