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

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

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

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

Beta-Test at UVM (Biology 204) We want to develop a new experimental protocol using a new organism (S.pombe) and a new experimental treatment (oxidative stress).

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

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?

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

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

Microarray Experiment The effect of a Hydrogen Peroxide (H 2 O 2 ) on gene expression in the yeast S. pombe. H 2 O 2 is one of the most powerful oxidizers known -- stronger than chlorine, chlorine dioxide, and potassium permanganate. And through catalysis, H 2 O 2 can be converted into hydroxyl radicals (. OH) with reactivity second only to fluorine.chlorinechlorine dioxidepotassium permanganate Grow the yeast and treat the control group with buffer (HBSS) and the treated group with buffer containing 0.5 mM H 2 O 2 Isolate RNA from the yeast grown in two different conditions, prepare target from it and use it on microarrays to see changes in gene expression

Expressed Genes = mRNA DNA (genes) messenger RNA Protein (effector molecules)

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

Experimental Design Choice of reference: Common (non- biologically relevant) reference, Non-treated, Wt Number of replicates: How many are needed? (How many are affordable?). Are the replicate results going to be averaged or treated independently? Is this a “fishing expedition” or a hypothesis- based experiment?

The steps of a microarray experiment:

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

RNA-DNA Hybridization probe sets on chip (DNA) (25 base oligonucleotides of known sequence) Targets (RNA)

Non-Hybridized Targets are Washed Away “probe sets” (oligos) Targets (fluorescently tagged) Non-bound ones are washed away

Human U133A DNA GeneChip - This GeneChip contains nearly 500,000 DNA oligos comprising 22,283 genes -The image on the left is a full scan of the U133A GeneChip while the image on the right is a 1000X zoom of a small area.

Why Use Yeast (S. pombe)?? easily manipulated in the laboratory simple eukaryote, unicellular rapid growth (doubling hours) non-pathogenic stable haploid and diploid states complete genome sequenced

E. coli ~ 1 x 3  m Yeast (S. pombe) ~ 12  m. Human ~ 1.7 m 1 chromosome 4 x 10 6 bp 3 chromosomes 12.5 X10 6 bp 23 chromosomes 3.3 x 10 9 bp ~ 4,377 genes ~ 25, 000 genes ~ 5004 genes

S. pombe Life Cycle

Yeast Microarray Experiment versus untreated H 2 O 2 treated Changes in gene expression? Which genes are up regulated? Which genes are down regulated? What do the results say about yeast biology? What parallels (if any) can we make to human biology?

Good Luck with the experiment! VGN Outreach Instructors: Pat Reed Scott Tighe Ahmad Chaudhry Janet Murray Tim Hunter