Large Scale Gene Expression with Large Scale Gene Expression with DNA MicroarraysDNA Microarrays

Vermont Genetics NetworkVermont Genetics Network

Microarray Outreach Microarray Outreach ProgramProgram

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 = mRNAExpressed 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 500-20,000)

• 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?What are Microarrays?

Why use 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?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 DesignExperimental 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 The steps of a microarray microarray experiment:experiment:

Creating TargetsCreating Targets

Reverse Transcriptase

in vitro transcription

mRNA

cDNA

cRNA

cDNA2nd Strand

1st Strand

RNA-DNA HybridizationRNA-DNA Hybridization

probe sets on chip(DNA)

(25 base oligonucleotides of known sequence)

Targets(RNA)

Non-Hybridized Targets are Washed AwayNon-Hybridized Targets are Washed Away

“probe sets” (oligos)

Targets(fluorescently tagged)

Non-bound ones are washed away

Why Use Yeast (Why Use Yeast (S. cerevisiaeS. cerevisiae)??)??

• easily manipulated in the laboratory

• simple eukaryote, unicellular

• rapid growth (doubling 1.5 - 2.5 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 cycleYeast Life cycle

S. cerevisiaeS. cerevisiae Genome Genome

• DNA ~ 60% A + T

• 16 chromosomes (haploid)

• chromosomes have centromeres and telomeres

• also have mitochondrial genome

• plasmid: 6 kb, 60-100 copies/cell

• entire genome has been sequenced

Yeast Genetic NomenclatureYeast 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 MetabolismGrowth and Metabolism

CARBON + NITROGEN + BIOTIN + MINERALS

+ O2 / no O2

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 ExperimentYeast 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?