1. Vermont Genetics Network Microarray Outreach Program Large Scale Gene Expression with DNA Microarrays

2. Vermont Genetics Network (VGN) Founded at the University of Vermont in 2001 through an NIH BRIN grant and renewed in 2005 through an NIH INBRE grant Purpose: Encourage biomedical research in Vermont Create a “network” of researchers and students Give outreach lectures to 4-year institutions Provide research grants to faculty and students Mentoring for students interested in research

3. VGN Microarray Outreach Program Develop microarray outreach module. Introduce microarray technology to VT colleges. Team of scientists to serve as instructors Tim Hunter, Pat Reed, Janet Murray, Scott Tighe

4. Why Gene Expression using Microarray? Up and coming modern technology- complicated Not affordable to all colleges Well adapted mini coarse with a “big picture” Can use many model organisms Employs large scale bioinfomatics-one of VGN’s goals

5. Desired Outcomes of Microarray module Learn new techniques that are state of the art Improve laboratory skills Build self confidence Creates new opportunities: Internships Job offers due to experience in new technology Build contact networks

6. Microarrays

7. Microarrays are simply small glass or silicon slides upon the surface of which are arrayed thousands of features (usually between 500 up to 7.5 million) Using a conventional hybridization process, the level of expression of genes is measured Microarrays are read using laser-based fluorescence scanners What are Microarrays?

8. Instrumentation Affymetrix GeneChip System 3000-7G Scanner 450 Fluidic Station 640 Hybridization Oven Affymetrix GeneChip $400 each $300,000

9. Arabidopsis ATH1 Genome Array This GeneChip contains 500,000 DNA oligos comprising 24,000 genes - The image on the left is a full scan of the GeneChip while the image on the right is a 1000X zoom of a small area.

10. Why use Microarrays? What genes are Present/Absent in a cell? What genes are Present/Absent in the experiment vs. control? Which genes have increased/decreased expression in experiment vs. control? Which genes have biological significance?

11. Microarray Applications Identify new genes implicated in disease progression and treatment response (90% of our genes have yet to be ascribed a function) Assess side-effects or drug reaction profiles Extract prognostic information, e.g. classify tumors based on hundreds of parameters rather than 2 or 3. Identify new drug targets and accelerate drug discovery and testing

12. Gene Discovery- –Assigning function to sequence –Discovery of disease genes and drug targets –Target validation Genotyping –Patient stratification (pharmacogenomics) –Adverse drug effects (ADE) Microbial ID Microarray Applications

13. 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. Patterns/clusters of expression are more predictive than looking at one or two prognostic markers – can figure out new pathways

14. The 2008 Marlboro Experiments What is your experiment What is your question or hypothesis What is your organism Will you be able to keep the study system stable and comparable How many duplicates/samples will be enough

15. Experimental Design Is this a “fishing expedition” or a hypothesis-based experiment? Choice of reference (control): Common reference, Non-treated, Wildtype As important as the experimental samples Number of replicates (required!!!): How many are needed ? How many are affordable? Pooling of samples???

17. Normal vs. Normal Normal vs. Treated UP Down Scatter plots reveal changes between samples

18. Laboratory Procedures and Considerations

19. DNA (genes) messenger RNA Protein (effector molecules) The Central Dogma [Expressed Genes = mRNA]

20. Consider the characteristics of your model organism!

21. E. coli ~ 1 x 3  m Yeast ~ 5  m dia. Human ~ 1.7 m 1 chromosome 4 x 10 6 bp 16 chromosomes 12 x 10 6 bp 23 chromosomes 3.3 x 10 9 bp ~ 4,377 genes ~ 30, 000 genes ~ 5,726 genes

22. Arabidopsis Characteristics…. How many chromosomes? What is expected RNA yields? What are the problems that investigators encounter routinely? What genes do you expect will change? Check the databases: http://www.arabidopsis.org/ Netaffx-Affymetrix

23. First step –Get good total RNA Five types of RNA –Ribosomal- [rRNA-5s, 5.8s 16s, 18s, 25s 28s] –Transfer- [tRNA]-protein synthesis] –Messager- [mRNA-Poly adenylated] –Micro- [miRNA-RNA control] –Small Nuclear- [snRNA] We need total RNA –We need the rRNA as an indicator of quality

24. Creating Targets Reverse Transcriptase in vitro transcription mRNA cDNA cRNA cDNA 2 nd Strand 1 st Strand GeneChip

25. The steps of a microarray experiment

26. Quality Control Is Very Important because microarray is very expensive! RNA purity and integrity Nanodrop spec Bioanalyzer cDNA synthesis efficiency Efficient cRNA synthesis, labeling, and fragmentation Target evaluation with Test Chips

27. RNA-DNA Hybridization, Staining, and Scanning Labeled sample Inject and Hyb 16hr Stain and scan Examine and Analyze…..

28. Data analysis is difficult and time consuming and can takes weeks and months due to the amount of data Data Analysis:

29. General Techniques and Guidelines for Extracting Total RNA

30. Why Total RNA? Not all transcripts have poly A tail RNA assessment is more determinative due to rRNA subunit peaks when running a gel or Bioanalyzer Recovery of special RNA’s such as MiRNA mRNA recovery kits also recover rRNA anyway

31. General RNA Handling Reagents and Equipment-Considerations: All reagents MUST be RNase-free Use gloves that are periodically treated with RNase Zap Perform as must work in a hood Biosafety Laminar flow PCR hood DO NOT use a fume hood Use aerosol resistent pipet tips Prepare all surfaces and pipets by treating with RNAse Zap All utensils [scissors, scalpels, tweezers] should be scrubbed clean, sprayed with RNase Zap, soaked in ETOH and flame sterilized before a surgery

32. Prepare daily aliquots of RNase-free water. I aliquot 10-20 tubes each week and discard half way through the day. Diethylpyrocarbonate [DEPC]-treated water is NOT an inhibitor for RNases, but rather DEPC is a chemical added to water to eliminate RNases. After autoclaving [or when purchased], no DEPC resides in the water. When opening and closing tubes, be careful not to bump the inner rim of your tubes. General RNA Handling

33. RNA Extraction Systems

34. RNA Isolation and Purification Systems-[Column-based] RNeasy Micro kit [74004] Small elution volumes 10-15ul for 10ug Good for FACS samples, LCM, or limited cell On-column DNase treatment RNeasy Mini Kit [74104] Standard Elution volume of 30-50ul for 100ug General use column On column DNase treatment Lipid or Fiber kits too RNeasy Midi and Maxi Kit Large elution volumes 150-800ul for 1mg of RNA USB Corp Prep-Easy Kit [78766] A knock off of the RNeasy Mini kit Elution volume of 30ul Cheaper than RNeasy and comes with DNase Zymo Corp Not recommended-poor recoveries in our hands

35. RNeasy and USB system are a 4M guanidine isothiocyanate [GCN] chaotopic salt system that strongly denatures RNases-no phenol RNA is precipitated with ethanol and bound to silica [Si-O-H], washed, and eluted with water. PROS CONS Easy to performWill not isolate MiRNA No precipitation rxnWill not isolate RNA <200bp Very clean RNALower yield than Trizol Can extract in RLT BufferLipids can interfere and inhibit DNase on the columnPoor storage integrity at -80 Compatible with FastPrepAbrasives can end up in final sample Compatible with Shedder column Heavy DNA contamination –bad [neutrophils, PMN] Salts may be left behind RNA Isolation and Purification Systems-[Column-based]

36. Silica Columns-how they work Once water is introduced, the electrostatic bond is “broken” and RNA is immediately released back into the “polar” water

37. Silica Column-based Applications [RNeasy]…continued Multiple applications of the same elution water increases yield Be sure not to “spin-out” your DNase before adding RW1 Do not spin with column with cap “open” as described in directions The final dry spin can be done for 3 minutes Some columns allow a final rinse with 80% ETOH to reduce salt carryover- Ask your tech support 260/280 ratios are often above 2.00 Can add 0.5ul of Superase ™ to collection tube if imparitive-Under special circumstances If using MinElute or MicroElute columns-be aware of the o-ring-it catches and retains liquid that can get into your final sample

38. Extracting RNA

39. Extracting RNA from Tissue- Types of homogenization Traditional mortor and pestle with LN2 Liquid Nitrogen is not always RNase-free Difficult to make sterile and RNase-free Poly-tron with new tips or RNase-free blades Not optimized for very small quantities Sometimes difficult to make RNase-free Mini motorized pestle With or without abrasive All RNase-free disposable Impactor [bio-pulverizer] French Press Biomasher columns

40. FastPrep System and Mini-bead beater Automated, fast, RNase-free Uses screw cap 2ml tubes, 15ml and 50ml and optional abrasive for homogenizing tough tissue Excellent for bacterial extractions Extracting RNA from Tissue- Types of homogenization Beware: Some beads are silica-based and can bind nucleic acids in the presence of binding buffers

41. Quality Control of RNA

42. Consider running an RNase-free gel –Look for gDNA –Look for rRNA bands We use the E-gel routinely –Fast, RNase-free agarose gel Quality Control of RNA

43. Measuring your RNA on the Nanodrop –Look carefully at the trace! –Can not distinguish DNA from RNA –Can not distinguish degraded RNA from “good” RNA –Quantitative interferences can lead to questionable downstream results Good RNA RNA prep with mostly gDNA from Neutrophils

44. Good vs Degraded RNA Thing are not always as they appear- These look great on the nanodrop… but…. are they? Quality Control of RNA

45. Quality Control of RNA ….expected yield? Expected yield is very important!!! Just getting “enough” RNA is not always ok Actively growing mammalian cells contain 1-10 pg/cell Calculate your expected RNA recovery If you are way below your expected yield than…. –Selectively recovered RNA from weak or apoptotic cells –Selectively recovered RNA from G0 or G2M ***Will significantly impact gene expression data*** We see apx 500 ng of RNA per 1mg of tissue

46. Problematic Nanodrop Traces Sample is NOT 183 ng/ul most likely much less 272 nm peak is skewing data 260 272 How will this affect downstream processes such as RT-qPCR if one assumes equal RNA input to a cDNA reaction?

47. Marlboro 2007….the fun has just begun

48. How do you use this thing?....

49. Oh, no…where’s my RNA?

50. Sites that have participated: Marlboro College St. Michael’s College Johnson State College Middlebury College Green Mountain College Norwich University Castleton State College Lyndon State College University of Vermont