Microarray analysis

INTRODUCTION
The large-scale genome sequencing effort and the
ability to immobilize thousands of DNA fragments on
coated glass slide or membrane, have led to the
development of microarray technology.
A microarray is a pattern of ssDNA probes which are
immobilized on a surface called a chip or a slide.
 Microarrays use hybridization to detect a specific
DNA or RNA in a sample.
 DNA microarray uses a million different probes,
fixed on a solid surface.
WHAT IS AN ARRAY

• An array is an orderly
arrangement of samples where
matching of known and
unknown DNA samples is done
based on base pairing rules.

• An array experiment makes use

of common assay systems such
as microplates or standard
blotting membranes.

Fig- Robotic arm with spotting slides

HISTORY

• Microarray technology evolved from Southern

blotting.
• The concept of microarrays was first proposed in
the late 1980s by Augenlicht and his colleagues.
• They spotted 4000 cDNA sequences on
nitrocellulose membrane and used radioactive
labeling to analyze differences in gene expression
patterns among different types of colon tumors in
various stages of malignancy.
PRINCIPLE

• The core principle behind

microarrays is hybridization
between two DNA strands.

• Fluorescent labeled target

sequences that bind to a
probe sequence generate a
signal that depends on the
strength of the hybridization
determined by the number of
paired bases.
Fig- Array hybridization
DNA MICROARRAY TECHNOLOGY
• DNA microarray technology may be defined as a
high-throughput and versatile technology used for
parallel gene expression analysis for thousands of
genes of known and unknown functions.
• Used for detection of polymorphisms and mutations
in genomic DNA
• A DNA microarray is a collection of microscopic
DNA spots on solid surface. Each spot contains
picomoles of a specific DNA sequence, known as
probes or reporters.
• Each identified sequenced gene on the glass, silicon
chips or nylon membrane corresponds to a fragment
of genomic DNA, cDNAs, PCR products or chemically
synthesized oligonucleotides of up to 70mers and
represents a single gene.

• Probe-target hybridization is usually detected and

quantified by detection of fluorophore, silver, or
chemiluminescence labeled targets to determine
relative abundance of nucleic acid sequences in the
target.
STEPS PERFORMING MICROARRAY ANALYSIS

• The principle of DNA microarray technology is

based on the fact that complementary sequences of
DNA can be used to hybridise, immobilised DNA
molecules.
• There are four major steps in performing a typical
microarray experiment.

Sample preparation Image acquisition

and Hybridisation Washing and
labeling Data analysis
SAMPLE PREPARATION AND LABELING
• Isolate a total RNA containing
mRNA that ideally represents a
quantitative copy of genes
expressed at the time of sample
collection.
• Preparation of cDNA from mRNA
using a reverse-transcriptase
enzyme.
• Short primer is required to initiate
cDNA synthesis.
• Each cDNA (Sample and Control) is
labelled with fluorescent cyanine
Fig- Sample labeling
ARRAY HYBRIDISATION

• Here, the labelled cDNA

(Sample and Control) are
mixed together.
• Purification of labelled
cDNA
• After purification, the mixed
labelled cDNA is
competitively hybridised
against denatured PCR
product or cDNA molecules
spotted on a glass slide.

Fig- Array Hybridisation

IMAGE ACQUISITION AND DATA ANALYSIS

• Slide is dried and scanned to

determine how much labelled
cDNA (probe) is bound to each
target spot.
• Hybridized target produces
emissions.
• Microarray software often uses
green spots on the microarray to
represent upregulated genes.
• Red to represent those genes that
are down regulated and yellow to
present in equal abundance

Fig- Gene chip showing different

type of color spots
 TYPES OF DNA MICROARRAY

1) Glass cDNA microarrays which involves the micro spotting of pre-

fabricated cDNA fragments on a glass slide.

2) High-density oligonucleotide microarrays often referred to as a

"chip" which involves in situ oligonucleotide synthesis.
 GLASS cDNA MICROARRAYS

• Glass cDNA microarrays was the

first type of DNA microarray
technology developed.
• It was pioneered by Patrick Brown
and his colleagues at Stanford
University.
• Produced by using a robotic
device which deposits (spots) a
nanoliter of DNA onto a coated
microscopic glass slide (50-150
µm in diameter) .

Fig- Contact printer with

robotic pins
MANUFACTURING OF GLASS cDNA MICROARRAY

Selection of the material to spot

onto the microscope glass
surface.

Preparation and purification of

DNA sequences representing
the gene of interest.

Spotting DNA solution onto

chemically modified glass slides
via a contact printing or inkjet
printing.
FIG- Spotting of slides
ADVANTAGES OF GLASS cDNA MICROARRAYS

• Advantages of Glass cDNA microarrays include their

relative affordability with a lower cost.
• Its accessibility requiring no specific equipment for
use such that hybridisation does not need specialized
equipment.
• Data capture can be carried out using equipment that
is very often already available in the laboratory.
DISADVANTAGES OF GLASS cDNA MICROARRAYS

• Glass cDNA microarray have a few disadvantages

such as intensive labour requirement for synthesizing,
purifying, and storing DNA solutions before
microarray fabrication.
• They may hybridise to spots designed to detect
transcript from a different gene.
IN SITU OLIGONUCLEOTIDE ARRAY

• Oligonucleotides are synthesized on the chip.

• Presently, the commercial versions of Affymetrix
Gene Chips hold up to 5,00,000 probes/sites in a
1.28-cm2 chip area.
• Due to such very high information content (genes)
they are finding widespread use in the
hybridisation-based detection and analysis of
mutations and polymorphisms, such as single
nucleotide polymorphisms.
 In situ light-directed oligonucleotide probe array synthesis.

• Light is directed through a

photolithographic mask to specific
areas of array surface.
• Activation of areas for chemical
coupling. Attachment of A nucleotide
containing photolabile protecting
group X (MeNPOC).
• Next light is Directed to a different
region of the array surface through a
new mask.
• Addition of 2nd building block T
containing a photolabile protecting
group X. This process is repeated until
the desired product is obtained.
Fig- Photolithography process
ADVANTAGES OF IN SITU OLIGONUCLEOTIDE
ARRAY FORMAT
• Advantages offered by the in situ oligonucleotide
array format include speed, specificity and
reproducibility.
DISADVANTAGES OF IN SITU OLIGONUCLEOTIDE ARRAY
FORMAT
• In situ oligonucleotide array formats tend to have expensive specialised
equipments e.g. to carry out the hybridisation, staining of label,
washing, and quantitation process.

• Short-sequences used on the array have decreased sensitivity/binding

compared with glass cDNA microarrays.
 Applications of Microarray Technology

MICROARRAY MICROARRAY
AS A GENE AS A
DISEASE DRUG TOXICOLOGICAL
EXPRESSION COMPARATIVE
PROFILING GENOMICS DIAGNOSIS DISCOVERY RESEARCH
TOOL TOOL
MICROARRAY AS A GENE EXPRESSION PROFILING TOOL

• The principle aim of using microarray technology as a gene

expression profiling tool is to answer some of the fundamental
questions in biology such as "when, where, and to what
magnitude genes of interest are expressed.

• Microarray analysis measure changes in the multigene

patterns of expression to better understand about regulatory
mechanisms and broader bioactivity functions of genes.
MICROARRAY AS A COMPARATIVE GENOMICS TOOL

• Microarray technology have widespread use in

comparative gene mutation analysis to analyse
genomic alterations such as sequence and single
nucleotide polymorphisms.

• In microbiology microarray gene mutation analysis is

directed to characterization of genetic differences
among microbial isolates, particularly closely related
species.
DISEASE DIAGNOSIS

• Different types of cancer have been classified on the

basis of the organs in which the tumors develop.

• Now, with the evolution of microarray technology, it

will be possible for the researchers to further classify
the types of cancer on the basis of the patterns of
gene activity in the tumor cells.
DRUG DISCOVERY

• Microarray technology has extensive application in

Pharmacogenomics.

• Comparative analysis of the genes from a diseased

and a normal cell will help the identification of the
biochemical constitution of the proteins synthesized
by the diseased genes.
TOXICOLOGICAL RESEARCH

• Microarray technology provides a robust platform for the

research of the impact of toxins on the cells and their passing
on to the progeny.
• Toxicogenomics establishes correlation between responses to
toxicants and the changes in the genetic profiles of the cells
exposed to such toxicants.
• The microarray permits researchers to examine thousands of
different genes in the same experiment and thus to obtain a
good understanding of the relative levels of expression
between different genes in an organism.