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

Microarray Experiment The effect of a chemical dimethyl sulfoxide (DMSO) on gene expression in yeast DMSO is an environmental contaminant from the paper industry and from pesticides Grow the yeast and treat one group with plain water (control group) and the other group with 10% DMSO 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)

Microarrays are simply small glass or silicon slides upon the surface of which are arrayed thousands of genes (usually between ,000) 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” 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?

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, or paired samples? 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

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

Why Use Yeast (S. cerevisiae)?? 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 ~ 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

Yeast Life cycle

S. cerevisiae Genome DNA ~ 60% A + T 16 chromosomes (haploid) chromosomes have centromeres and telomeres also have mitochondrial genome plasmid: 6 kb, copies/cell entire genome has been sequenced

Yeast Genetic Nomenclature genes names are 3-letters + a number genes names are derived from phenotype of the mutant, i.e. ste 3 mutants have sterile phenotype gene names are written in italics or underlined wild type alleles: Uppercase STE 3 mutant alleles: lowercase ste 3 Yeast strain : NRRL Y12362

Growth and Metabolism CARBON + NITROGEN + BIOTIN + MINERALS + O 2 / no O 2 Prototroph: requires no additional nutrients (can grow in minimal media) Auxotroph: requires additional nutrients from the environment or media NRRL Y12362 is auxotrophic for leucine, tryptophan, uracil, adenine, and histidine YPD is a rich media; therefore, NRRL Y12362 can get these nutrients from the environment

Yeast Microarray Experiment versus untreated DMSO 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?