Transcript Day1VGN-Microarray-CSC2011ppt

Microarray: Global Transcriptional
Expression Profiling
Vermont Genetics Network
Microarray Outreach
Program
What is an IDeA State?
The National Institutes of Health
(NIH) Institutional Development
Award Program (IDeA) was
established in 1993 to broaden the
geographic distribution of NIH
funding for biomedical and
behavioral research.
-Increase Competiveness
-Build capacity
-Advance research through
animal models
-Foster research through
technologies
-Develop informatics approaches to
support research
-Strengthen the research workforce
-Maximize partnerships
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
• Encourage biomedical research in Vermont
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Create a “network” of researchers and students
Give outreach lectures to 4-year institutions
Provide research grants to junior faculty and students
Mentoring for students interested in research
Core Facility Development and Support
Building Statewide Network Through Outreach
Activities: Inter-Institutional
2001: 5 BPI Partners
-Develop culture of research
Lyndon State
-Funding for Faculty
Development and
Undergraduate Student
Research
-Infrastructure Support
Green Mountain College
-Career Development for
Undergraduate Students
-Access to UVM Core
Facilities and Expertise
Marlboro College
(Outreach only)
2010 submission: 7 BPI
partners
INBRE: Vermont Genetics Network
Core Facilities
Microarray Core
-Established in 2002
Bioinformatics Core
-Established in 2002
Outreach Core
-Established in 2003
Proteomics Core
– Established in 2006
VGN Outreach Modules:
• Microarray
First Delivery in 2003
• Bioinformatics
First Delivery in 2006
• Proteomics
First Delivery in 2009
VGN Microarray Outreach Program
• Develop microarray outreach module
• Provide necessary resources (footlocker)
• Introduce microarray technology to VT
colleges.
• Team of scientists to serve as instructors
Tim Hunter, Kara Pivarski
Heather Driscoll, Janet Murray
Sites that have participated in Microarray
Outreach:
UVM
St. Michael’s College
Johnson State College
Middlebury College
Green Mountain College
Norwich University
Castleton State College
Lyndon State College
Marlboro College
Bates College (ME)
What are Microarrays?
• Microarrays are simply small glass or silicon
slides upon the surface of which are arrayed
thousands of genes (usually between 500-20,000)
– 11 probe sets/transcript, 3’ biased
• 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?
Instrumentation
Affymetrix GeneChip System
3000-7G Scanner
(2)-450 Fluidic Station
640 Hybridization Oven
Affymetrix GeneChip
$400 each
$300,000
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
• Differential Expression
– Ctrl vs Treated (Exposed)
• Gene Discovery
– Assigning function to sequence
– Discovery of disease genes and drug targets
– Target validation
• Genotyping
– Patient stratification (pharmacogenomics)
– CNV, SNP
• Microbial ID
Arabidopsis ATH1 Genome Array
This GeneChip contains 500,000 DNA oligos comprising 24,000 genes
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The image on the left is
a full scan of the
GeneChip while the
image on the right is a
1000X zoom of a small
area.
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
Is this a “fishing expedition” or a hypothesis-based experiment?
Choice of reference (control):
Common reference, Non-treated, Wildtype
As important as the experimental samples
Number of replicates (required!!!):
How many are needed ?
How many are affordable?
Pooling of samples???
Central Dogma:
DNA (genes)
messenger RNA
Protein (effector molecules)
The steps of an
“Eberwine
approached “
microarray
experiment:
The 2008 Castleton Experiments
• What is your experiment
• What is your question or hypothesis
• What is your organism
• Will you be able to keep the study system stable and
comparable
• How many duplicates/samples will be enough
Questions
• What traits/genes enable A. lyrata to grow well in such
extreme conditions?
– Sugars and sugar alcohols act like antifreeze
– Adaptation of photosynthesis to cold conditions, low light
• Are plants surviving in warmer conditions by growing only in
wet, relatively low light environments?
• Can plants survive and adapt to rapidly warming (and drying)
environments?
Hypothesis
• Arabidopsis lyrata is historically adapted to periglacial conditions
– Relatively cold, wet, sometimes saline soil conditions under
mostly cloudy (i.e., lower average light) conditions
• Predictions:
– Plants should have higher fitness levels in periglacial locations
than warmer locations (e.g., Norway vs. Ireland)
• Greater plant size, higher reproductive output (flowers and
seeds)
– Plant populations should be larger in periglacial locations
– Given repeated historical advance and retreat of glaciers:
• A. lyrata should tolerate different substrate (soil) types
• A. lyrata may have physiological and morphological
plasticity to facilitate acclimation and adaptation to variable
conditions
Microarray Outreach Outcomes:
The coordination and delivery of the microarray modules to each Vermont
college is only the beginning of our relationship with the college’s faculty and
students.
Core :
-Collaborations
-Facility Usage
-Networking Throughout
the State
-Interactions with Students
Faculty:
-Gaining Expertise
-Curriculum Development
-Granting Opportunities
-Collaborations
-More Scientific
Colleague
Undergraduate
Students:
-Learn new techniques
that are state of the art
-Improve laboratory skills
-Build self confidence
-New opportunities:
-Internships
-Job offers due to
micorarray exp
-Build contact networks
Castleton Outcomes…These and More???
Good Luck with the experiment!
VGN Outreach Instructors:
Tim Hunter
Janet Murray
Kara Pivasrki
Heather Driscoll