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History
cDNA microarrays have evolved from Southern blots, withcDNA microarrays have evolved from Southern blots, with
clone libraries gridded out on nylon membrane filtersclone libraries gridded out on nylon membrane filters
being an important and still widely used intermediate.being an important and still widely used intermediate.
Things took off with the introduction of non-porous solidThings took off with the introduction of non-porous solid
supports, such as glass - these permitted miniaturization -supports, such as glass - these permitted miniaturization -
and fluorescence based detection. Currently, about 20,000and fluorescence based detection. Currently, about 20,000
cDNAs can be spotted onto a microscope slide.cDNAs can be spotted onto a microscope slide.
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Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
THE PROCESSTHE PROCESS
Building the Chip:
MASSIVE PCR PCR PURIFICATION
and PREPARATION
PREPARING
SLIDES
PRINTING
Preparing RNA:
CELL CULTURE
AND HARVEST
RNA ISOLATION
cDNA PRODUCTION
Hybing the Chip:
POST PROCESSING
ARRAY HYBRIDIZATION
PROBE LABELING
DATA ANALYSIS
4. 10/04/16 4Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
cDNA clones
(probes)
PCR product amplification
purification
printing
microarray Hybridise target
to microarray
mRNA target)
excitation
laser 1laser 2
emission
scanning
analysis
overlay images and normalise
0.1nl/spot
5. 10/04/16 5Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Preparing RNA:
RNA ISOLATION
cDNA PRODUCTION
Designing experiments to profile conditions/perturbations/
mutations and carefully controlled growth conditions
RNA yield and purity are determined by system. PolyA isolation is preferable
but total RNA is useable. Two RNA samples are hybridized/chip.
Single strand synthesis or amplification of RNA can be performed.
cDNA production includes incorporation of Aminoallyl-dUTP.
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Preparing RNA:
RNA ISOLATION
cDNA PRODUCTION
Designing experiments to profile conditions/perturbations/
mutations and carefully controlled growth conditions
RNA yield and purity are determined by system. PolyA isolation is preferable
but total RNA is useable. Two RNA samples are hybridized/chip.
Single strand synthesis or amplification of RNA can be performed.
cDNA production includes incorporation of Aminoallyl-dUTP.
Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Preparing RNA:
RNA ISOLATION
cDNA PRODUCTION
Designing experiments to obtain conditions necessary to
induce changes in the plaque to cause its rupture
RNA yield and purity are determined by system.
Single strand synthesis or amplification of RNA can be performed.
OBTAINING THE
TARGET TISSUE
6. 10/04/16 6Department of Statistics, University of California, Berkeley, and
Hybing the Chip:
ARRAY HYBRIDIZATION
PROBE LABELING
DATA ANALYSIS
Cy3 and Cy5 RNA samples are simultaneously
hybridized to chip. Hybs are performed for 5-12 hours
and then chips are washed.
Two RNA samples are labelled with Cy3 or
Cy5 monofunctional dyes via a chemical
coupling to AA-dUTP. Samples are purified
using a PCR cleanup kit.
Ratio measurements are determined via
quantification of 532 nm and 635 nm
emission values. Data are uploaded to the
appropriate database where statistical and
other analyses can then be performed.
7. 10/04/16 7Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
Biological
Question
Sample
preparationMicroarray
Life Cycle
Data Analysis
& Modeling
Microarray
Reaction
Microarray
Detection
aken from Schena & Davis
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Our Aim
• We intend to induce rupture of atherosclerotic plaque in
apo-e mice by injecting several drugs, which we hope will
cause rupture of the plaque by altering the hemodynamic
system and raising the oxidative stress and alter the
composition of the plaque.
• After achieving this goal we will perform gene- array
study on these specimens and will try to find out which
genes are expressed more in plaques which are ruptured or
are prone to rupture, by comparing our results at the
histopathology lab which will define the structural details
of such vulnerable plaques.
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DNA Sequence Synthesis
We are going to design a genearray slide based onWe are going to design a genearray slide based on
oligonucleotide sequences that we will deriveoligonucleotide sequences that we will derive
from the genes that we are interested in.from the genes that we are interested in.
To do this, we will proceed as follows:To do this, we will proceed as follows:
First we will gather the cDNA or mRNAFirst we will gather the cDNA or mRNA
sequences of the desired genes from Entrez.sequences of the desired genes from Entrez.
Then we will pickup a sequence of genes from thatThen we will pickup a sequence of genes from that
particular gene and by using Primer Premierparticular gene and by using Primer Premier
program we will design a primer to use as aprogram we will design a primer to use as a
hybridization probe.hybridization probe.
12. 10/04/16 12Department of Statistics, University of California, Berkeley, and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research,
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We will order a probe to be made by a company that willWe will order a probe to be made by a company that will
make that on a commercial basis.make that on a commercial basis.
The micro array-chip, we will make at the lab byThe micro array-chip, we will make at the lab by
synthesizing cDNA from mRNA, that we isolated.synthesizing cDNA from mRNA, that we isolated.
We will obtain the tissue samples from the mice, accordingWe will obtain the tissue samples from the mice, according
to our study design, we will purify the mRNA accordingto our study design, we will purify the mRNA according
the specific protocol.the specific protocol.
mRNA from the target will be converted into cDNA, andmRNA from the target will be converted into cDNA, and
allowed to hybridiz on the microarray chip that weallowed to hybridiz on the microarray chip that we
designeddesigned
The emission of colors will be captured by the scanner,The emission of colors will be captured by the scanner,
and the results will be analysed depending upon theirand the results will be analysed depending upon their
intensity and presence and absence of colorintensity and presence and absence of color
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• Drugs being used to
induce rupture of
atherosclerotic plaque
in apo-e mice
