DNA Microarray
Rajal Debnath
Sushil Kumar
M.Sc Biotechnology, CMRIMS
DNA Microarrays
A small 1 square centimeter chip
thats divided into thousands of
squares.
Each square contains many copies of
a single gene.
Originally developed by Patrick Brown
at the Stanford University School of
Medicine to determine which genes
are involved in yeast cell sporulation.
Sushil Kumar,Rajal Debnath
DNA Microarrays
Enable one to simultaneously measure the
level of activity of up to 60,000 genes
Powerful technology for biological
exploration
In particular, the amount of mRNA for each
gene in a given sample (or a pair of samples)
is measured
Sushil Kumar,Rajal Debnath
Basic Idea
Every cell of the body contains a full set
of chromosomes and identical genes
Only a fraction of these genes are
turned on, and "expressed
Results in unique properties of each cell
Sushil Kumar,Rajal Debnath
Basic Idea
Central dogma
Complementary hybridization
DNA
mRNA
cDNA
ATCGTAGCTAGCGATCG
A
TAGCATCGATCGCAAGC
T
Sushil Kumar,Rajal Debnath
Basic Idea
A simple concept: Dot Blot + Northern
Reverse the hybridization - put the
probes on the filter and label the bulk
RNA
Make probes for lots of genes - a
massively parallel experiment
Make it tiny, so you dont need much
RNA from experimental cells
Make quantitative measurements
Sushil Kumar,Rajal Debnath
Technique
Isolation of the DNA sequences
Reverse transcription of the gene
Hybridization
Scanning
Sushil Kumar,Rajal Debnath
Sushil Kumar,Rajal Debnath
Sushil Kumar,Rajal Debnath
Instruments
Arrayer
Scanner : Laser Confocal Microscope
Sushil Kumar,Rajal Debnath
Arrayer
Sushil Kumar,Rajal Debnath
Scanner
GenPix 4000
Sushil Kumar,Rajal Debnath
The sample printer
Sushil Kumar,Rajal Debnath
Procedure
Biological
Question
Sample
Preparation
Data Analysis
& Modeling
Microarray
Detection
Microarray
Reaction
Sushil Kumar,Rajal Debnath
Procedure
Preparation
Target DNA (reference and test samples)
Slides
Reaction(Droplet or Pin Spotting)
Hybridization
Scanning
Analysis
Arrayer
Hardware
Scanner
Software
Image processing
Data mining
Modeling
Sushil Kumar,Rajal Debnath
excitation
cDNA clones
(probes)
cDNA arrays
laser 2
in summary
PCR product amplification
purification
printing
scanning
laser 1
emission
mRNA target)
overlay images and normali
0.1nl/spot
microarray
Hybridise
target to
microarra
y
Sushil Kumar,Rajal Debnath
analysis
Affymetrix processing steps
Sample RNA isolation
cDNA synthesis
Biotin-labeled cRNA synthesis
cRNA fragmentation
Quality control
procedures
Gel electrophoresis,
OD
Gel electrophoresis
Gel electrophoresis, OD
Gel electrophoresis
Hybridization to array
Array wash and stain
Array scanning
Examination of the intensity of the
image
Examination of chip quality
indicators, and control probe
Sushil Kumar,Rajal Debnath
sets
Image analysis
procedure
Sushil Kumar,Rajal Debnath
Sushil Kumar,Rajal Debnath
Sushil Kumar,Rajal Debnath
Sushil Kumar,Rajal Debnath
Sushil Kumar,Rajal Debnath
Sushil Kumar,Rajal Debnath
Sushil Kumar,Rajal Debnath
Sushil Kumar,Rajal Debnath
Sushil Kumar,Rajal Debnath
Sushil Kumar,Rajal Debnath
Sushil Kumar,Rajal Debnath
Sushil Kumar,Rajal Debnath
Sushil Kumar,Rajal Debnath
Sushil Kumar,Rajal Debnath
Sushil Kumar,Rajal Debnath
The outcome
Sushil Kumar,Rajal Debnath
Inference
GREEN represents Control DNA, where either DNA or
cDNA derived from normal tissue is hybridized to the
target DNA.
RED represents Sample DNA, where either DNA or cDNA
is derived from diseased tissue hybridized to the target
DNA
YELLOW represents a combination of Control and
Sample DNA, where both hybridized equally to the target
DNA
BLACK represents areas where neither the Control nor
Sample DNA hybridized to the target DNA
Sushil Kumar,Rajal Debnath
Data
Quantification
Sushil Kumar,Rajal Debnath
Before labeling
Sample 1
Sample 2
Array 2
Array 1
Sushil Kumar,Rajal Debnath
Before Hybridization
Sample 1
Sample 2
Array 2
Array 1
Sushil Kumar,Rajal Debnath
After Hybridization
Array 2
Array 1
Sushil Kumar,Rajal Debnath
Quantification
Array 2
Array 1
Sushil Kumar,Rajal Debnath
Quantification
Sushil Kumar,Rajal Debnath
Microarray Data Analysis
Data is vast
Impossible to analyse the data
visually
Special softwares are required to
analyse the microarray data .
Sushil Kumar,Rajal Debnath
Softwares
Expression Profiler
(http://www.ebi.ac.uk/expressionprofiler)
GEO (Gene Expression Omnibus)
(http://www.ncbi.nlm.nih.gov/geo)
GeneSpring
Sushil Kumar,Rajal Debnath
example
Sushil Kumar,Rajal Debnath
GEO at the NCBI
Sushil Kumar,Rajal Debnath
GeneSpring
Sushil Kumar,Rajal Debnath
Applications
Gene expression studies
Gene function for cell state change in
various conditions (clustering,
classification)
Disease diagnosis (classification)
Inferring regulatory networks
Pathogen analysis
Sushil Kumar,Rajal Debnath
Applications
Drug Discovery
identify appropriate molecular targets for
therapeutic intervention
monitor changes in gene expression in
response to drug treatments
Targeted Drug Treatment
Sushil Kumar,Rajal Debnath
Cluster by
color
difference
Sushil Kumar,Rajal Debnath
Applications
How to sort samples into two classes
based on gene expression data
Cancer vs. normal
Cancer sub-types
(benign vs. malignant)
Responds well to drug vs. poor
response
(i.e. tamoxifen for breast cancer)
Sushil Kumar,Rajal Debnath
Conclusion
Important in Functional Genomics.
Take a list of "interesting" genes and find
their biological relationships.
Gene lists may come from
significance/classfication analysis of
microarrays, proteomics, or other highthroughput methods
Rapid and efficient method for faster &
accurate results.
Sushil Kumar,Rajal Debnath
Questions or Comments
Sushil Kumar,Rajal Debnath
Thank you!!!
Sushil Kumar,Rajal Debnath
