Transcript Microarray - Clemson University

Introduce to
Microarray
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Evolution & Industrialization
1989: First Affymetrix Genechip Prototype
1994: First Commercial Affymetrix Genechip
1994- First cDNAs arrays were developed at Stanford University.
1994: First Commercial Scanner-Affymetrix
1996- Commercialization of arrays
1997-Genome-wide Expression Monitoring in S. cerevisiae
Why use DNA Microarrays for
Expression Analysis?
• Conventional expression analysis only allows
the study of the expression of a single gene in a
single experiment
• The highly parallel nature of microarrays allows
the simultaneous study of the expression of
thousands or even tens of thousands of different
genes in a single experiment
• Microarrays allow researchers to undertake
global expression analysis that is not feasible
with conventional techniques
What Microarrays detect?
• 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
The 6 steps of a DNA
microarray experiment
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 (cont.)
4. Image acquisition (scanning) and
quantification (signal intensity to numbers)
5. Database building, filtering and
normalization
6. Statistical analysis and data mining
Different Types of DNA
Microarrays
How DNA
sequences
are laid down
Spotting
Photolithography
(Affymetrix)
type of DNA
sequences
cDNA
(Complete
sequences)
Oligonucleotides
Affymetrix Microarrays
Involves Fluorescently tagged cRNA
• One chip per sample
• One for control
• One for each experiment
Spotted Microarrays
Involves two dyes/one chip
• Red dye
• Green dye
• Control and experiment on same chip
Production of Affymetrix Arrays
• Choice of probes
– 25mer oligonucleotides
– 11-16 per gene
– 3’ biased
– Dispersed
– Chosen to minimize cross-hybridization
• Computer algorithms are used to design
photolithographic masks for use in
manufacturing
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Each gene is represented on the probe
array by multiple probe pairs
Each probe pair consists of a perfect
match and a mismatch oligonucleotide
Match and mismatch
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The exact match is a section of the mRNA sequence you wish to probe for
The mismatch is identical except for one base difference from it’s exact
match counterpart, and is used to calculate a background.
There are typically 11 “probe pairs” scattered around the chip- called a
probe set.
By combining the expression values for a probe set, a value for the
expression of mRNA can be found.
Mismatch probe:
Perfect match probe:
Target sequence:
ATGCTGTACAATCGCTTGATACTGG
ATGCTGTACAATAGCTTGATACTGG
ATGCTGTACAATAGCTTGATACTGG
Why do we have mismatch
probes?
• Mismatch probes (MM) are trying to detect
background.
• The mismatch probes are supposed to
detect things that are close but not an
exact match.
• It is assumed that these things also bind to
the perfect match (PM), erroneously.
Production of Affymetrix Arrays:
Photolithographic Synthesis
Creating Targets
mRNA
Reverse Transcriptase
cDNA
in vitro transcription
cRNA
Target
RNA-DNA Hybridization
Targets
RNA
probe sets
DNA
(25 base oligonucleotides of known sequence)
Non-Hybridized Targets are Washed
Away
Targets
(fluorescently tagged)
“probe sets” (oligo’s)
Non-bound ones are washed away
Custom GeneChips
Affymetrix offers over 120 prokaryotic arrays that are
manufactured by Nimblegen Inc.
Custom GeneChips are also available for both
Eukaryotic and Prokaryotic systems.
Spotted Arrays
• Robotically spotted cDNAs or
Oligonucleotides
• Printed on Nylon, Plastic or Glass surface
– DNA probes prepared in 384-well plates
– Glass slides bought commercially or prepared in
house
• Arrays are spotted with a commercial arrayer
• Replicates
• Controls
Microarray of
thousands of
genes on a
glass slide
steel
Spotted arrays
spotting pin
chemically modified slides
384 well source
plate
1 nanolitre spots
90-120 um diameter
Building the chip
Arrayed Library
(96 or 384-well plates of
bacterial glycerol stocks)
Spot as microarray
on glass slides
PCR amplification
Directly from colonies with
SP6-T7 primers in 96-well
plates
Consolidate into
384-well plates
Expression profiling with DNA
microarrays
cDNA “B”
Cy3 labeled
cDNA “A”
Cy5 labeled
Laser 1
Hybridization
Laser 2
Scanning
+
Analysis
Image Capture
Spotted Arrays
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Homemade
Tailored
Cheaper?????
Maximum 24,000
features per array
• Prone to variability
Affymetrix Arrays
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Commercially available
“Off the rack”
More expensive?????
Maximum 500,000
features per array
• Less variability
Oligonucleotide
• Greatly reduced
cross-hybridization
• Uniform Tm
• Requires knowledge
of gene sequences
• More expensive???
cDNA
• Cross-hybridization
possible
• Non-uniform Tm
• No gene sequence
knowledge required
• Less expensive
Microarray Experiments Animation
•http://www.bio.davidson.edu/courses/genomics/chip/chip.html
Biological
Question
Data
Analysis &
Modelling
Microarray
Life Cycle
Sample
Preparation
Microarray
Detection
Microarray
Reaction
W.W.W resources
• Complete guide to “microarraying”
http://cmgm.stanford.edu/pbrown/mguide/
• http://www.microarrays.org
– Parts and assembly instructions for printer and
scanner;
– Protocols for sample prep;
– Software;
– Forum, etc.
• Animation:
http://www.bio.davidson.edu/courses/genomics/chip/chip.html