Transcript Day1-UVM-2ndvisit-Pombe

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).
What are Microarrays?
• 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”
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
Microarray Applications
• 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 Experiment
• The effect of a Hydrogen Peroxide (H2O2) on gene expression in
the yeast S. pombe.
• H2O2 is one of the most powerful oxidizers known -- stronger
than chlorine, chlorine dioxide, and potassium permanganate.
And through catalysis, H2O2 can be converted into hydroxyl
radicals (.OH) with reactivity second only to fluorine.
• Grow the yeast and treat the control group with buffer (HBSS)
and the treated group with buffer containing 0.5 mM H2O2
• 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 (nonbiologically 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 hypothesisbased experiment?
The steps of a
microarray
experiment:
Creating Targets
mRNA
Reverse Transcriptase
1st Strand
cDNA
2nd Strand
cDNA
in vitro transcription
cRNA
GeneChip
RNA-DNA Hybridization
Targets
(RNA)
probe sets on chip
(DNA)
(25 base oligonucleotides of known sequence)
Non-Hybridized Targets are Washed
Away
Targets
(fluorescently tagged)
“probe sets” (oligos)
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 1.5 - 2.5
hours)
• non-pathogenic
• stable haploid and diploid states
• complete genome sequenced
E. coli
Yeast (S. pombe)
Human
~ 1 x 3 m
~ 12 m .
~ 1.7 m
1 chromosome
3 chromosomes
23 chromosomes
4 x 10 6 bp
12.5 X10 6 bp
3.3 x 10 9 bp
~ 4,377 genes
~ 5004 genes
~ 25, 000 genes
S. pombe Life Cycle
http://www-rcf.usc.edu/~forsburg/main.html#cycle
Yeast Microarray Experiment
untreated
Changes in gene expression?
Which genes are up regulated?
versus
Which genes are down
regulated?
What do the results say about
yeast biology?
H2O2 treated
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