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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. Purpose: Encourage biomedical research in Vermont - PowerPoint PPT Presentation
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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.
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 • Introduce microarray technology to VT colleges. • Develop microarray outreach module. • Team of scientists to serve as instructors
Ahmad Chaudhry, Rebecca Guy, Tim Hunter, Brian McElhinney, Pat Reed
Kathy Seiler, Scott Tighe
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
Creating TargetsCreating Targets
Reverse Transcriptase
PCR amplification of DNA
in vitro transcription
mRNA
cDNA
cRNA
cDNAMore
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
The 6 steps of a DNA microarray The 6 steps of a DNA microarray experiment (1-3)experiment (1-3)
1. Manufacturing of the microarray
2. Experimental design and choice of reference: what to compare to what?
3. Target preparation (labeling) and hybridization
The 6 steps of a microarray experiment (4-6)The 6 steps of a microarray experiment (4-6)
4. Image acquisition (scanning) and quantification (signal intensity to numbers)
5. Database building, filtering and normalization
6. Statistical analysis and data mining
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?
Why Use Yeast ??Why Use Yeast ??
• 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
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.
Our strain, NRRL Y-12632 (or ATCC 18824) is a wild type, prototrophic, Matα strain isolated from brewing yeast.
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?