MICROARRAY TECHNIQUE

ARUN CHACKO
PhD SCHOLAR
PLANT BREEDING & GENETICS
 An array
• Orderly arrangement of samples where
matching of known and unknown DNA
samples is done based on base pairing rules.
• A DNA microarray (DNA chip or biochip) is a
collection of microscopic DNA spots attached to a
solid surface.
• An array experiment makes use of common
assay systems such as microplates or standard
blotting membranes.
 History of Microarray
• 1965 : Gillespie and Spiegelman described
methods for DNA blotting hybridization, in
which DNA immobilized on a membrane can
bind to complementary RNA or DNA strand
through specific hybridization.

• 1975: Southern blotting developed by E. M.

Southern
• 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.
• 1982: RNA was isolated from normal and
cancer tissue of mice, cDNA synthesized,
cloned on E. Coli and 378 colonies were
arrayed.
• 1991: Scientists at the California-based
biotech company Affymetrix produce the first
DNA chips.
• 1995: Quantitative monitoring of gene
expression patterns with a complementary
DNA microarray.
• 1995:microarrays for gene expression
profiling was done using complete eukaryotic
genome (Saccharomyces cerevisiae) on a
microarray chip.
 The main applications of microarrays include:

Comparative genome analysis (Healthy and infected

cell)
Functional genome analysis (to describe interactions
between genes at different time point)
Developing knowledge of gene function
Discovery of drugs
Diagnostics and genetic engineering
Toxicological research (Toxicogenomics)
 Principle of Microarray technique
• mRNA : carries the genetic information from
the cell nucleus to the cytoplasm for protein
synthesis. These mRNAs synthesize the
corresponding protein by translation.
• Indirectly by assessing the various mRNAs,
we can assess the genetic information or the
gene expression thus helps in the
understanding of various processes behind
every altered genetic expression.
• The principle behind microarrays is
hybridization between two DNA strands
 Major steps in Microarray
1. Preparation of microarray
2. Preparation of labelled probes
3. Hybridization
4. Scanning, imaging and data analysis
 Methods for constructing the arrays:
PROBE TYPE ADVANTAGES DISADVANTAGES
PCR Products Inexpensive Handling problems
Hard to design to avoid
cross- hybridization
Unequal amplification

Oligos Can be designed for many Expensive

criteria
Easy to handle Normalized
concentrations

Affymetrix GeneChip High quality data Expensive

Standardized arrays Arrays available for limited
Fast to set up number of species
Multiple probes per
gene
 Microspotting Techniques

• Conventional methods can be used to produce the

sequences (oligonucleotides), and these can then be
printed directly onto the microscope slide (which is
first overlaid with a coating that is positively charged).
 Piezoelectric Printing

•First used by AGILENT

•This is a non-contact process.
•Minute volumes of reagents are delivered to
defined locations on the slide similar to
‘ink-jet’ printing methods.
•A biochemical sample is loaded into a
miniature nozzle equipped with a piezoelectric
fitting (rectangles) and an electrical current is
used to expel a precise amount of liquid from
the jet onto the substrate.
After the first jetting step,
the jet is washed and a
second sample is loaded and
deposited to an adjacent
address. A repeated series
of cycles with multiple jets
enables rapid microarray
production.
 Photolithography

• This makes use of semiconductor technologies.

• This ‘in situ’ fabrication technique was developed by
Affymetrix, and is used to produce their Gene Chips.
• A mercury lamp is used, activates DNA bases.
 The photolithographic method
• Treat substrate with chemically protected “linker”
molecules, creating rectangular array.
• Selectively expose array sites to light deprotects
exposed molecules, activating further synthesis .
• Flush chip surface with solution of protected
A,C,G,T.
• Binding occurs at previously deprotected sites.
• Repeat steps 2&3 until desired probes are
synthesized.
• The mask only allows light to pass to specific
features on the chip.
•A chip modified with photolabile protecting groups is selectively
activated for DNA synthesis by shining light through a photomask.
•The chip is then flooded with a photoprotected DNA base, resulting in
spatially defined coupling on the chip surface.
•A second photomask is used to deprotect defined regions of the chip.
•Repeated deprotection and coupling cycles enable the preparation of
high- density oligonucleotide microarrays.
• Principle of microarray technique
• Preparation of microarray
– Microspotting technique
– Piezoelectric printing
– Photolithography
 DNA Microarray Technology
• 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 and represents a single
gene.

• Probe-target hybridization is usually detected and

quantified by detection of fluorophore, silver, or
chemiluminescence labelled targets to determine
relative abundance of nucleic acid sequences in the
target.
• The principle of DNA microarray technology
is based on the fact that complementary
sequences of DNA can be used to hybridise,
immobilised DNA molecules.

Sample
Preparation Washing Image Data Analysis
and labelling acquisition
Sample preparation

Cy 3 Cy 5
 Array Hybridisation
Labelled cDNA mixed together

Purification

the mixed labelled cDNA is competitively hybridised

against denatured PCR product
- cDNA molecules spotted on a glass slide
• Slide is dried and scanned to determine how
much labelled cDNA (probe) is bound to each
target spot.
• Hybridized target produces emissions.
Upregulated genes

Downregulated genes

Equal abundance
 Types of DNA Microarray/ DNA Chips

• cDNA based microarray

• Oligonucleotide based microarray

 cDNA based microarray
• The first type of DNA microarray technology
developed.
• This type of chips are prepared by using
cDNA, it is called cDNA chips or cDNA
microarray or probe DNA.
• The cDNAs are amplified by using PCR.
• These are immobilized on a solid support
made up of nylon filtre of glass slide.
• 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).
 Sample Preparation
 mRNA has been extracted from the cells or tissues
under study, it is converted into DNA by the use of
the reverse transcriptase enzyme.

 During this reaction, the DNA is labelled by the

incorporation of fluorescent or radioactive
nucleotides into the DNA.

 The two samples are labelled using two different

fluorescent dyes - say, red or green. The most
common dyes in use are Cy3 (Green) and Cy5
(Red).
 Oligonucleotide based microarray

• Often referred to as a "chip" which involves

in situ oligonucleotide synthesis.
• Gene chip (DNA chip, Affymetrix chip)
• Oligonucleotide (20~80-mer oligos) is
synthesized in situ (on-chip).
• Developed at Affymetrix, Inc. , under the
GeneChip® trademark
AFFYMETRIX MICROARRAY
 Affymetrix Chip
• Each gene has 16 – 20 pairs of probes synthesized on the
chip.
• Each pairs of probes have two oligonucleotide.
• Perfect match (PM, reference seq) ATG…C…TGC (20-25
bases).
• Mismatch (MM, one base change) ATG… T …TGC
• A MM oligo is identical to a PM oligo except that the
middle nucleotide (13 th of 25) is intentionally replaced by
its complementary nucleotide .
• The scanned result for a given gene is the average
differences between PM and MM signals, over probes.
A Probe Set for Measuring Expression Level of a
Particular Gene probe pair gene sequence
• The black features represent no intensity (no RNA hybridized
to the respective probe in the feature).
• The intensity level from lowest to highest by colour is:
Dark blue -> Blue -> Light Blue -> Green -> Yellow ->
Orange -> Red - > White .
• More intensity means more RNA bound to a specific feature,
which basically means the gene was expressed at a higher
level.
Affymetrix GeneChip experiment
 Affymetrix GeneChip experiment
• Labelled cRNA randomly fragmented in to pieces anywhere
from 30 to 400 base pairs in length.
• The fragmented, Biotin-labeled cRNA is added to the array
• Anywhere on the array where a cRNA fragment and a probe
are complimentary, the cRNA hybridizes to the probes in the
feature.
• The array is then washed to remove any cRNA that is not
stuck to an array then stained with the fluorescent molecule
that sticks to Biotin (Cy5 conjugated to streptavidin).
• Lastly, the entire array is scanned with a laser and the
information is kept in a computer for quantitative analysis of
what genes were expressed and at what approximate level.
Procedure
 Analysis of DNA microarray
• Bioconductor, an open source and open
development software project for the analysis
of genomic data primarily based on the R
programming language, contains a number of
program packages for microarray data
analyses and is arguably the most
comprehensive resource for such applications.
(Gentleman et al., 2004)
 Advantages of DNA microarray
• To study the behaviour of many genes simultaneously.
• The technique is very fast: there can be as many as 150
copies of an array of 12,000 genes printed in only 1 day.

• DNA microarray technology is relatively cheap to use:

• the initial cost of constructing an arrayer is
approximately $60,000;
• after this, the cost per copy of a microarray is
small, usually less than $100.
• The technique of DNA microarrays is very user-friendly:
• the technique is neither radioactive nor toxic
• the microscope slide is a convenient base for
the technique
• arrays are cheap and easily replaced
• A major advantage of DNA microarrays is that
information about the sequence of the DNA is not
required to construct and use the DNA microarrays.
 Limitations
• As well as the cost of robotics to perform the
technique, there may be technical limitations.
• The technique of DNA microarrays is currently limited
not by the number of probes on an array, but by the
resolution of the scanner used.
• Too much data all at once. Can take quite a while
to analyze all the results.
• The results may be too complex to interpret
• The results are not always reproducible
• The results are not always quantitative enough
• The technology is still too expensive
Microarray in Genomics
• The main application of genomic microarrays is
represented by gene expression profiling.
• Basically, two types of genomic microarrays are
available:
• wide genome and focused arrays.
• Wide-genome arrays are designed to bear on them as
many genes as possible.
• Currently Affymetrix HU133 plus v.2 gene chips
have around 47 000 genes or (ESTs) on them.
• Focused arrays are designed to bear few
tens/hundreds of genes of interest.
• Gene expression studies: data sets can vary greatly
in size. The data heterogeneity is where the big
challenge lies for the scientist, and a crucial role is
thus played by bioinformatics and biostatistics.
• Softwares: Microarray Suite, GeneSpring, Partek
Pro.
• All software packages are equipped with visualization
tools, such as self-organizing maps, hierarchical
clustering, principal component analysis, relevance
networks, etc.
• Self-organizing maps: genes are plotted on a
two-dimensional graph based on their
expression level across all samples.
• Thus, groups of genes with a similar
expression pattern will have a similar trend
within the graph.
Hierarchical clustering analyses:
• Genes are plotted against samples with a dendrogram,
which is sort of a mock phylogenetic tree, whose
branches connect genes related by a similar expression
pattern: the shorter the branch, the stronger the
correlation.
• The dendrogram is connected with its branches to the
clustering map, where genes are represented by squares
colored in green/red or blue/yellow, based on their
differential expression level (up- or down-regulation),
which define specific molecular fingerprints.
• Even though visualization tools can give a
comprehensive overview of the whole study
performed, they are usually derived from the
application of filters resulting in lists of at least
hundreds of genes that might be difficult to interpret
and make a sense of, without additional statistical
analyses.

• Once a specific set of genes is identified as the

significant one, validation studies are required in
order to confirm gene expression profile
observations.
Such validation should be preferentially done by
• RT-PCR, quantitative PCR (Taqman),
• Northern and Western blotting,
• RNA protection assay, flow cytometry,
• mice knockout models.

All targeted to reconfirm previous gene expression

profiling data.
• cDNA microarrays containing ~9,000 unigenes was
used to identify 486 salt responsive expressed
sequence tags(ESTs) (representing ~450 unigenes) in
shoots of the highly salt-tolerant rice variety, Nona
Bokra (Oryza sativa L. ssp. Indica pv. Nona).
• This study identified a large number of candidate
functional genes that appear to be involved in salt
tolerance and further examination of these genes may
enable the molecular basis of salt tolerance to be
elucidated.
• The rice BiostarP-100s cDNA microarray (United
Gene Holdings,Ltd., PRC), containing 10,060
sequences representing ~9000 unigenes including
novel, known and control genes, was used to identify
salt-regulated genes.
• Gene expression was examined at three time points
after salt treatment (20min, 3h and 24h)
corresponding to early transient, intermediate and late
regulation.
• The significantly regulated genes at each time point
were selected for cluster analysis and for inclusion in
the salt-induced-microarray (SIM).
• The fluorescent signatures were scanned and
captured using a ScanArray4000 Standard
Biochip Scanning System.
• Data were analyzed using the GenePix Pro 3.0
software.
• The utilized genes were amplified by
polymerase chain reaction (PCR) of the
appropriate rice cDNA clones using T3 and T7
primers.
• After the resulting products were purified and
confirmed by direct sequencing, the fragments
were printed on slides using an OmniGrid
printer.
 Comparison of salt response gene expression in salt-stressed
Nona and IR28 plants using hierarchical cluster analysis

• Red : up-regulated genes

• Green: down-regulated
genes;
• Black: un-regulated
genes
• Blanks: missing data.
 Advantages of genomic microarray
• One shot genome wide expression analysis.
• Rapid comparison between two states (control/diseased,
untretead/treated, and wild type/knockout).
• Exploration of new biological systems in a hypotheses
generating rather than hypotheses testing fashion.
• Identification of markers to elucidate molecular mechanisms
(signatures) underlying biological events and diseases.
• Rapid molecular disease classification for more accurate
molecular diagnostic, prognostic and targeted treatment.
 Disadvantages
• Restricted access to the technology (experiments still
exprehensive to perform)
• Not yet approved as diagnostic tool by regulatory bodies
• Not a stand alone technique (need validation/confirmation
tests)
• Skilled technical personnel needed (including
biostatistician/bioinformatician for data analysis)
• Data derived from different platforms difficult to compare
• Data comparability difficult from one array version to the next
• Data obtained only partially used and published
• Data repositories and data sharing still not fully implemented
• Ethical and legal issues when dealing with patient samples
 PROTEIN MICROARRAY
• Protein microarrays are miniaturized and parallelized
array technology approaches for protein–protein
interactions analysis and protein profiling.

• Typically, thousands of proteins are printed and

immobilized on functionalized glass slides, which
that can be simultaneously studied and analyzed in a
HT fashion, thereby offering a high potential for
characterizing the biology of a given cell of interest.
• The first report of using protein arrays for protein–
protein interaction, ligand binding and biochemical
investigations was by MacBeath and Schreiber in
2000.
• The success of each microarray-based screening
heavily depends on the library construction and
microarray fabrication.
Three key areas of protein characterizations:
1. functional annotation,
2. substrate fingerprinting,
3. ligand/inhibitor binding.
• The major challenge as compared to DNA
microarray is that the need to maintain the
structural integrity and physicochemical
properties of proteins, derived from its complexity
& variability (e.g. post-translational modifications,
etc.).
 Classification of Protein Microarray
• Target microarrays,
• Reverse Phase arrays
• in situ expressed arrays.
 Detection techniques for Protein microarray
Conventional fluorescence labeling : the use cyanine dyes
(e.g.: Cy3 and Cy5)
•Target protein array
•NAPPA protein array (Nucleic Acids Programmable Protein Arrays )
•Reverse phase microarray

Detection for suspension array technology

•Flow cytometry
•Magnetic bead based detection
•Quantum dots
•Gold Nano particles

Label free techniques

•Surface Plasmon Resonance

•Microcantilevers and Atomic force microscopy