Microarray: Global Transcriptional Expression Profiling

1. Microarray: Global Transcriptional Expression Profiling Vermont Genetics Network Microarray Outreach Program

2. What is an IDeA State? The National Institutes of Health (NIH) Institutional Development Award Program (IDeA) was established in 1993 to broaden the geographic distribution of NIH funding for biomedical and behavioral research. -Increase Competiveness -Build capacity -Advance research through animal models -Foster research through technologies -Develop informatics approaches to support research -Strengthen the research workforce -Maximize partnerships

3. 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 • Encourage biomedical research in Vermont • Create a “network” of researchers and students • Give outreach lectures to 4-year institutions • Provide research grants to junior faculty and students • Mentoring for students interested in research • Core Facility Development and Support

4. Building Statewide Network Through Outreach Activities: Inter-Institutional • 2001: 5 BPI Partners • -Develop culture of research • -Funding for Faculty Development and Undergraduate Student Research • -Infrastructure Support • -Career Development for Undergraduate Students • -Access to UVM Core Facilities and Expertise • 2010 submission: 7 BPI partners Lyndon State Green Mountain College Marlboro College (Outreach only)

5. INBRE: Vermont Genetics Network Core Facilities • Microarray Core • -Established in 2002 • Bioinformatics Core • -Established in 2002 • Outreach Core • -Established in 2003 • Proteomics Core • Established in 2006

6. VGN Outreach Modules: • Microarray First Delivery in 2003 • Bioinformatics First Delivery in 2006 • Proteomics First Delivery in 2009

7. VGN Microarray Outreach Program • Develop microarray outreach module • Provide necessary resources (footlocker) • Introduce microarray technology to VT colleges. • Team of scientists to serve as instructors • Tim Hunter, Kara Pivarski • Heather Driscoll, Janet Murray

8. Sites that have participated in Microarray Outreach: UVM St. Michael’s College Johnson State College Middlebury College Green Mountain College Norwich University Castleton State College Lyndon State College Marlboro CollegeBates College (ME)

9. What are Microarrays? • Microarrays are simply small glass or silicon slides upon the surface of which are arrayed thousands of genes (usually between 500-20,000) • 11 probe sets/transcript, 3’ biased • Via a conventional DNA hybridization process, the level of expression/activity of genes is measured • Data are read using laser-activated fluorescence readers • The process is “ultra-high throughput”

10. What are Microarrays? Instrumentation Affymetrix GeneChip System 3000-7G Scanner (2)-450 Fluidic Station 640 Hybridization Oven Affymetrix GeneChip $400 each $300,000

11. 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?

12. Microarray Applications • Differential Expression • Ctrl vs Treated (Exposed) • Gene Discovery • Assigning function to sequence • Discovery of disease genes and drug targets • Target validation • Genotyping • Patient stratification (pharmacogenomics) • CNV, SNP • Microbial ID

13. 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.

14. 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

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???

16. Central Dogma: DNA (genes) messenger RNA Protein (effector molecules)

17. The steps of an “Eberwine approached “microarray experiment:

18. The 2008 Castleton 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

19. Questions • What traits/genes enable A. lyrata to grow well in such extreme conditions? • Sugars and sugar alcohols act like antifreeze • Adaptation of photosynthesis to cold conditions, low light • Are plants surviving in warmer conditions by growing only in wet, relatively low light environments? • Can plants survive and adapt to rapidly warming (and drying) environments?

20. Hypothesis • Arabidopsis lyrata is historically adapted to periglacial conditions • Relatively cold, wet, sometimes saline soil conditions under mostly cloudy (i.e., lower average light) conditions • Predictions: • Plants should have higher fitness levels in periglacial locations than warmer locations (e.g., Norway vs. Ireland) • Greater plant size, higher reproductive output (flowers and seeds) • Plant populations should be larger in periglacial locations • Given repeated historical advance and retreat of glaciers: • A. lyrata should tolerate different substrate (soil) types • A. lyrata may have physiological and morphological plasticity to facilitate acclimation and adaptation to variable conditions

21. Microarray Outreach Outcomes:The coordination and delivery of the microarray modules to each Vermont college is only the beginning of our relationship with the college’s faculty and students. Core : Faculty: Undergraduate Students: -Gaining Expertise -Curriculum Development -Granting Opportunities -Collaborations -More Scientific Colleague • -Learn new techniques that are state of the art • -Improve laboratory skills • -Build self confidence • -New opportunities: • -Internships • -Job offers due to • micorarray exp • -Build contact networks -Collaborations -Facility Usage -Networking Throughout the State -Interactions with Students Castleton Outcomes…These and More???

22. Good Luck with the experiment! VGN Outreach Instructors: Tim Hunter Janet Murray Kara Pivasrki Heather Driscoll