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

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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

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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