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Microarray Technology 273
CHAPTER
12 Microarray Technology
Microarray analysis has emerged in the last few years as a flexible method for analyzing large
numbers of nucleic acid fragments in parallel. Its origins can be traced to several different disci-
plines and techniques. Microarrays can be seen as a continued development of molecular biology
hybridization methods, as an extension of the use of fluorescence microscopy in cell biology, as
well as a diagnostic assay using capture to solid surface as a way to reduce the amount of analytes
needed. The convergence of ideas and principles utilized in these fields, together with technological
advancements in preparing miniaturized collections of nucleic acids on solid supports, have all
contributed to the emergence of microarray and microchip technologies. This technology promises
to monitor the whole genome on a single chip so that researchers can have a better picture of the
interactions among thousands of genes simultaneously.
Keywords: Microarrays, differential gene expression, reverse northern, hybridization, oligonucle-

otide arrays, cDNA arrays.
INTRODUCTION
Gene expression is a highly complex and tightly regulated process allowing a cell to respond
dynamically both to environmental stimuli and to its own changing needs. Each cell of an organism
usually contains the same set of genes, however there are significant differences in their activation
and control. Only a fraction of these genes are expressed in the form of proteins at a particular
time and confer unique properties to each cell type. The expression of a single gene can be detected
by Northern hybridization where the total RNA of different samples are blotted on a nylon mem-
brane and the expression of a gene of interest is detected by using probes of that particular gene
of interest. Single gene expression can also be detected by dot blot technique where the total RNA
of different samples (up to 96 samples) are immobilized on nylon membrane and expression of a
particular gene in all the samples detected by probe of that particular gene of interest. In the post-
genome sequencing era, a large number of gene sequences are known for different organisms,
hence there is a need to understand the change that occurring at transcriptome level, i.e., change
in the overall transcripts in a cell with respect to time or under different stress conditions. However,
the study of transcriptome needs a suitable experimental design and high-throughput instruments.
Many techniques are available for the study of transcriptome such as Reverse Northern Blotting
(RNB), Serial Analysis of Gene Expression (SAGE), subtractive hybridization (SH) or microarray.
In this chapter, we have focused on the principles and procedures of microarray.
274 Biotechnology in Medicine and Agriculture
DNA microarray is defined as an orderly arrangement of DNA fragments representing the genes
of an organism and requires specialized robotics and imaging equipment. It is also known as DNA
chip, biochip and gene array where thousands of different known genes are spotted on a suitable
support and the spot sizes are typically less than 200 microns in diameter. This technique was
originally designed to measure gene expression levels, but now it has revolutionized functional
genomic analysis. The chronological order of microarray development is summarized in Table 12.1.
Table 12.1 Chronological order of microarray development.
Year Brief description of the activity

1953 Watson and Crick described DNA double helix and their denaturation
1961 Marmur and Doty described DNA renaturation or molecular hybridization
1965 Gillespie and Spiegelman measured the interaction between an RNA molecule and the DNA
from which it was transcribed
1966 Khorana and coworkers demonstrated synthesis of complex nucleic acids.
1969 Jones and Robertson discovered in situ hybridization
1971 Kleppe provided the primers needed for the polymerase chain reaction (PCR) Engvall and
Perlman developed ELISA
1975 Grunstein and Hogness described colony hybridization
1977 Benton and Davis described plaque hybridization
1979 Kafatos introduced dot blot
Wallace introduced synthetic oligonucleotides as hybridization probes
1987 Wan, Fortuna and Furmanski described tissue microarray technique
1990 Ink-jet spotting methods for in situ synthesis of 60-mer oligo spots on glass slides
1991 Photolithographic printing (Affymetrix) developed by Fodor and colleagues
1992 Ward and coworkers introduced multicolor fluorescent labeling techniques in FISH
1992 Kallioniemi and coworkers termed comparative genomic hybridisation (CGH)
1994 First cDNA collections were developed at Stanford University (this was first step toward
microarrays)
Hoheisel and coworkers used multiple libraries for genome analysis
Hoheisel and coworkers replaced manual procedures by robotics
1995 Quantitative cDNA microarray
1996 Commercialization of oligonucleotide arrays (GeneChipTm by Affymetrix)
1997 Genome-wide expression analysis in S. cerevisiae (yeast)
1998 Kononen and coworkers developed tissue microarray technology
1999 Bulyk and coworkers described DNAprotein interactions in a microarray format
Mendoza and coworkers developed high-throughput microarray-based ELISA.
2000 Cancer signatures detected
Ciphergen Biosystems Inc. established protein chip array technology
de Wildt and coworkers first used antibody arrays to study antibody-antigen interactions
Afanassiev and coworkers describe protein microarrays on glass slides coated with an
agarose film
2001 Zhu and coworkers first used protein microarray to study protein-protein interactions in
yeast
2003 Clinical application of microarray
Rubina and coworkers describe protein microarrays on glass slides coated with polyacry-
lamide
Ge developed a universal protein array system (UPA) for quantitative detection of interac-
tions of protein with protein, DNA, RNA and ligand.
Contd.
Rauch and coworkers redefined CGH as molecular karyotyping

Angenendt and coworkers developed Multiple Spotting Technology (MIST)
2004 Whole human genome analysis by single microarray
Kersten and coworkers describe protein microarrays on glass slides coated with nitrocel-
lulose
There are different types of microarrays comprising different types of molecules such as
oligonucleotides, cDNAs, clones, PCR products, polypeptides, antibodies and others or tissue
sections immobilized at discrete ordered micrometer-to-millimeter-sized locations on the surface of
a porous or nonporous insoluble solid support. A microarray works by exploiting the ability of a
given mRNA molecule to hybridize specifically to the DNA template from which it originated. For
global analysis of gene expression, i.e., for transcriptome analysis, high density microarrays have
been developed. Newly annotated genes and novel genes can be analyzed by DNA microarray
analysis. A specific location is assigned to each DNA fragment representing a gene on the array.
The supports are usually glass microscope slides, silicon chips or nylon membranes. Over 30,000
spots can be placed on one slide through the use of robotic spotters. The spots themselves can
be DNA, cDNA, or oligonucleotides. The DNA can be printed or synthesized directly onto the
support. Fluorescently labeled DNA or RNA in the sample act as target, which will hybridize to
the complementary spots (probes) on the array. After this, these fluorescent tags are excited by
laser of wavelengths 635 nm (for Cy5), 532 nm (for Cy3). Hybridized DNA are identified as
glowing spots on the array by exposing the microarray to a scanner, while the spots that do not
hybridize will not be visible under the scanner. The microscope and camera work together to create
a digital image of the array. The computer program then creates a table that contains the ratios of
the intensity of red-to-green fluorescence for every spot on the array (Figure 12.1). In the following
text, we describe the basic steps of the making of microarray and the process of hybridization.
CONCEPTS AND PRINCIPLES

Despite the variety of technical solutions that have been developed for performing microarray
analysis, all are miniaturized hybridization assays for studying thousands of nucleic acid fragments
simultaneously. All microarray systems share the following key components:
the array, which contains immobilized nucleic acid sequences, or targets.
one or more labelled samples or probes that are hybridized with the microarray.
a detection system that quantitates the hybridization signal.
BASIC STEPS IN MICROARRAY TECHNOLOGY

Printing of Microarray
Probe samples are spotted (or printed) on a microscopic glass slide (coated with polylysine) with
the help of a microarrayer (or spotter). The polylysine coating is done to enhance binding of DNA/
RNA to the plate through electrostatic interactions. Polylysine, which is not fixed to DNA, is
blocked in order to avoid target binding. Besides glass slides, specially coated plastic films can also
be used as solid support. The spotting process is performed inside a dust- and vibration-free
chamber. Evaporation of the samples is further avoided by maintaining high humidity in the
Figure 12.1 Basic steps of microarray Technology: Probes are amplified by PCR and printed on
solid support. Total RNA samples extracted from treated and control cells/tissues
(Sample A and B) are reverse transcribed and labelled with either Cy3 (green) or
Cy5 (red) fluorescent dyes. The samples are combined and hybridised to the
microarray. After that washing was done to remove non-hybridized target, laser
scanning is carried out and the emissions measured in each colour channel.
Specialised software is used to measure intensity ratios to each spot, which are
then exported for statistical analysis to identify differentially expressed genes.
chamber. After printing, slides are left at room temperature for 24 h for efficient coupling of printed
cDNA. Finally, dried slides are put in a beaker for washing with ethanol followed by air-drying,
and stored at room temperature till further use. Prior to hybridization, DNA is denatured to obtain
a single strand DNA.
Slide preparation
Microarray slide preparation requires a suitable support so that the probes can be immobilized
efficiently. Fabrication is the process of spotting the probes on a suitable support. The support
on which the probes have to be spotted should be impermeable to solvent to avoid swelling and
shrinking during the experiment. Nitrocellulose membrane provides an excellent support for this
purpose as the pores of the membrane provide a larger total surface for binding.
However, reducing the size of spots beyond certain limits or controlling their size and shape on
a porous membrane is not possible. Thus, glass or plastic supports are preferred over membrane
support because first, these supports have dimensional stability and rigidity, whereas membranes
swell in solvent and tend to shrink and distort when dried. Secondly, nucleic acids form a mono-
layer saturating the surface on glass or plastic and hence the amount of attached DNA is consistent
from one region of the array to another. Thirdly, as they are in a monolayer on the surface, the
nucleic acids are favorably placed to take part in hybridization reactions. Fourthly, interactions with
the solution phase are much faster, because molecules do not have to diffuse into and out of the
pores. It has been estimated that the amount of material deposited on the surface of the substrate
forming monolayer is equivalent to 10 pmol/mm2. Therefore, currently, over 50,000 cDNA probes
can be spotted onto a 25 ´ 75 mm slide by robotic printing. Thus, the choice of support is extremely
critical determining the success of an experiment.
In this regard, either the presynthesized probes can be spotted or can also be synthesized in situ
on solid support by high throughput technology, which is described in the following text:
Presynthesized Probes: In this case, the presynthesized probes are covalently crosslinked
to the glass slides containing poly-L-lysine using ultraviolet irradiation. The method of
application of probes onto the glass slide is a precisely computer-controlled process where
a head carrying a pin or pen device is used to pick up small drops of solution containing
presynthesized probes from the multiwell plates followed by spotting them on to the surface
of glass slide.
In Situ Synthesis of Probes: Here, the different probes are directly synthesized on the
slide by the process of coupling of nucleotides, which, in turn, is a multistep process.
Probes can be synthesized either by using ink-jet fabrication, or flow channels or by light
directed fabrication.
(a) Fabrication by Ink-Jet Printers: This technique is based on the principle of ink-jet
printers, where the firing solutions of nucleotide reagents are spotted onto the glass
surface according to the information of probe sequences feeded in the computer.
Computer software is used for moving the pens and substrate as well as for printing
four colors for delivering precursors for four different bases. Thus, any set of oligo-
nucleotides can be made and known sequences can thus be placed at any position in
the array.
(b) Flow Channels and Cells: In this process, the precursors for the four bases, A, C,
G, T are introduced through channels to make 4 broad stripes of the mononucleotides
on a square plate. A second set of four nucleotides are laid down in four narrower stripes
within each of the monomers to create 16 stripes of dinucleotides. This process is
repeated until the oligonucleotides have reached half of probes final length. At this point,
the plate is turned 90o and the whole process is repeated. This method is particularly
used for making arrays either to those comprising all oligonucleotides of a given length
or those comprising all the complements of a target of known sequence. The dimen-
sions of such arrays are determined by the width of the stripes.
(c) Fabrication by Photolithography: In this technique of fabrication, the oligonucle-
otides are synthesized at specific positions on a glass surface by irradiation. Irradiation
of glass surface is done by using light of wavelength 365nm. For the addition of bases,
photolithographic masks are used for specifically synthesizing probes on a very small
area of glass surface. Light is passed through the mask where nucleotide has to be
added. When the light is passed through a specific areas on mask, the protecting group
on the 5' hydroxyl group of the previously added nucleotide gets activated. The surface
is then flooded with the coupling agent for the next base and the process continues till
the desired length of the probe is synthesized. This method has the advantage that any
sequence can be synthesized at any position in a random fashion within a small area.
Target preparation
Target is a sample for which the transcriptome analysis has to be performed. For the microarray
experiment, target sample has to be labeled with fluorescent dyes. mRNA of target sample is reverse
transcribed using reverse transcriptase and dNTP mixture containing amino-allyl dCTP into cDNA.
Amino-allyl nucleotide is very reactive for coupling with fluorescent dye, cyanine (Cy). Labeling
is performed with two dyes Cy3 (green dye) and Cy5 (red dye). One sample is labeled with Cy3,
whereas the other sample is labeled with Cy5 for the identification of differentially expressed genes.
Hybridization
Both green and red labelled cDNA (targets) are mixed together and put on the matrix of spotted
single strand DNA (probes). The chip is then incubated overnight for hybridization at 60oC under
highly humid conditions. At this temperature, DNA strands encounter the complementary strands
of the probes on the slide and create double stranded DNA i.e., fluorescent DNA hybridize on the
spotted ones. Double stranded DNA thus formed has one unlabelled and other labeled strand. To
obtain high quality data, effective hybridization of target is essential. Commercially available cover
slips with raised Teflon edging for full contact with arrayed probes (Lifter Slips; Erie Scientific,
Portsmouth, NH, USA), hybridization chambers for submerging microarray in water of set tem-
perature (Corning, NY, USA), hybridization solution (Clontech, Palo Alto, CA, USA) etc. are used
for effective hybridization. The hybridization rate is increased if the hybridization solution is in
motion over the surface of the array i.e., placing the array in a rotating cylinder.
Slide scanning
After hybridization and washing, microarrays are scanned at two different wavelengths corre-
sponding to the absorbance of red and green dye. The signals are analyzed by passing a beam of
laser through the microarray slide that excites each spot on the plate and the fluorescent emissions
are gathered through a photo-multiplicator tube (PMT) coupled with a confocal microscope. The
spot will appear either red or green if the hybridization is stronger with one of the samples. In
contrast, the spot on the microarray will appear to be yellow if the intensities of binding of two
dyes to target samples are equal. Microscopic detection and quantification of fluorescence images
provide the basic array data, but variance is increased by background fluorescence, dust, and spot-
to-spot and array-to-array differences in signal intensity. Due to this, complex normalization and
correction routines must be applied to the resulting data.
Data analysis
Now there are two images from the same slide corresponding to the two dyes from which we have
to calculate the number of DNA molecules in each experimental condition. For any spot on the slide,
we measure the signal intensities in the green dye emission wavelength and the red dye emission
wavelength. If the mRNA amount used for hybridization is proportional to the amount of fluores-
cent DNA fixed onto the plate, one can directly calculate the red/green fluorescence ratio. Ratio
greater than 1 (red on the image) shows that the gene expression is greater in the sample 2, while
ratio smaller than 1 (green on the image) indicates that gene expression is greater in sample 1. These
differences in expression can be visualized and interpret using commercially available software (for
example, array plot). The signals intensities of the spots are correlated with the concentrations of
target mRNA samples. Data mining is conducted by using statistical programs and algorithms to
determine whether the gene of interest is up-regulated, down-regulated, or unchanged. Data is then
organized using a database and gene annotation can be performed with gene ontology (GO)
analysis, clustering analysis and pathways analysis and networking of genes can also be predicted.
To adjust the data for systemic non-biological effects arising from technical variation(s) and
measurement, error normalization is essential. The aim is to remove the effect of noise from the
data while still maintaining the ability to detect significantly differentially expressed genes. Still,
there is no universally accepted method of normalization, but the intensity dependent normalization
i.e., local weighted regression is accepted most.
Expression profile clustering

Clustering of expression profiles obtained through arrays can be done at the end of microarray
analysis. Genes sharing same expression profile gradually form clusters during phylogenetic analy-
sis. Other complex techniques such as principal component analysis or neuronal networks are also
now being used for this purpose. Microarray data is visually presented in a two-dimensional table
or heat-map, each cell of which uses a simple color code to represent the relative transcript
expression of a single gene under each of a defined set of experimental conditions. The vertical axis
identifies each gene in the collection, whereas the horizontal axis displays each condition or time-
point in a time series analysis. Final data is presented in the form of hierarchical clustering, where
each column represents the microarray data from one experiment and each row a specific gene.
Rigorous experimental design and statistical analysis, together with adequate replication, is critical
to draw broad conclusions about the biology of a system based on microarray data. Mostly
microarray experiments are designed with only one categorical factor, so paired t-test is used for
statistical analysis, but for multiple categorical factors (time and genotype) we require analysis of
variance (ANOVA) based methods. B-statistic is an estimate of the odds that the gene is differen-
tially expressed. Commercially available software packages for normalization, statistical analysis
and visualization are Cyber-T, SAM, BRB-Array Tools (http://linus.nci.nih.gov/BRB-ArrayTools),

QVALUE, Focus, statistical language R, etc.
Microarray Data Management

As a single chip may contain 30,000 spots of target DNA, there is necessity of uniform system
that manages and provides a disbursement point for microarray data. As the proficiency in gen-
erating data is overcoming the capacity for storing and analyzing, this data requires standardization
of storage and sharing. For public use and dissemination of gene expression data, NCBI has
launched the Gene Expression Omnibus (GEO). It has an expression data repository and online
resources for the storage and retrieval of gene expression data from any organism or artificial
source. GEO will aid in the study of functional genomics by storing vast amounts of data on gene
expression profiles derived from multiple experiments using varied criteria and conditions. GEO
facilitates cross-validation of data obtained using different techniques and technologies and helps
to set standards for further gene expression studies.
Microarray Markup Language (MAML) is developed by Microarray Gene Expression Database
(MGED) and is the first attempt to provide a standard platform for submitting and analyzing the
enormous amounts of microarray expression data generated around the world. The goal is to
facilitate the adoption of standards for DNA-array experiment annotation and data representation,
as well as the introduction of standard experimental controls and data normalization methods and
also to facilitate the establishment of gene expression data repositories, the comparability of gene
expression data from different sources, and data analysis software. MAML is independent experi-
mental platform and provides a framework for describing experiments done on all types of DNA
arrays. MAML provides a format that allows analysis of data obtained not only from any existing
microarray platforms but also many of the possible future variants, including protein arrays.
Additional data set from different experiments available in the Barley Base repository for cereal Gene
Chip data (http://barleybase.org/), ArrayExpress (http://www.ebi.ac.uk/arrayexpress/) and
Microarray Gene Expression Data Society (http://www.mged.org/) provides an invaluable resource
for the scientific community. Different website links related to gene sequences, different tech-
niques, various companies, laboratories and bioinformatics tools are given in Table 12.2.
Table 12.2 List of some representative websites related to microarray.

Types Websites
Gene Sequences
Cancer Genome Anatomy Project (CGAP) ncbi.nlm.nih.gov/CGAP
CGAP EST database ncbi.nlm.nih.gov/dbEST/index.html
Ensemble resources Ensembl.org
ESTs identified by the Institute of Genomic tigr.org
Research (TIGR)
IMAGE consortium bio.llnl.gov/bbrp/image/image.html
Incyte incyte.com
Purchasing UniGene clones image.llnl.gov/image/htmlidistributors.shtml
Research Genetics http://www.resgen.com/index.php3
UniGene resources ncbi.nlm.nih.gov/UniGene
Contd.
Microarray laboratories
Albert Einstein College of Medicine sequence:aecom.yu.edu/bioinf/funcgenomic.html
Ed Southern bioch.ox.ac.uk/rgroups/rgroupslist.asp
Institute of Cancer Research wyvis.icr.ac.uk
National Human Genome Research Institute nhgri.nih.gov/DIR/LCG/15K/HTML
(NHGRI)
Pat Brown laboratory cmgm.stanford.edu/pbrown/index.html
Vivian Cheung Laboratory w95vcl.neuro.chop.edu/vcheung
Company links
Affymetrix affymetrix.com
BD Biosciences bdbiosciences.com
BioRobotics biorobotics.co.uk
Chroma chroma.mbt.washington.edu
Ciphergen Biosystems Inc ciphergen.com
Genometrix genometrix.com
General Scanning genescan.com
Gene Logic genelogic,.com
Hyseq hyseq.com
Incyte incyte.com
Luminex® Corporation luminexcorp.com
Molecular Dynamics mdyn.com
Molecular Staging, Inc. molecularstaging.com
Protometrix, Inc. protometrix.com
Pierce Biotechnology Inc. searchlightonline.com
RayBiotech Inc. raybiotech.com
Schleicher & Schuell Bioscience schleicher-schuell.de/bioscience
SIGMA-ALDRICH Co. sigmaaldrich.com
Zeptosens AG zeptosens.com
Zyomyx Inc. zyomyx.com
Bioinformatics
ArrayExpress ebi.ac.uk/arrayexpress
ArrayTrack (NCTRs Center for fda.gov/nctr/science/centers/toxicoinformatics/
Toxicoinformatics Array Track) ArrayTrack
CEBS (Chemical Effects in Biological Systems) cebs.niehs.nih.gov
CIBEX (Center for Information Biology) cibex.nig.ac.jp
GEO (Gene Expression Omnibus) ncbi.nlm.nih.gov/geo
NHGRI Tissue Microarray Project nhgri.nih.gov/DIR/CGB/TMA
Stanford Link Page Genomewww4.Stanford.EDU/MicroArray/
SMD/restech.html
Tox-MIAMExpress (Toxicogenomics MIAMExpress) ebi.ac.uk/tox-miamexpress
UCLA Tissue Array Core Facility genetic.ucla.edu/tissuearray
Whitehead Institute http://www.wi.mit.edu/nap/features/
nap_feature_sabatiniappt.html
Techniques
BioArray Software Environment BASE base.thep.lu.se
Bio-IT World bio-itworld.com
ESF Programme on Integrated Approaches for functionalgenomics.org.uk/sections/resources/
Functional Genomics protein_arrays.htm
Genomics Proteomics genomicsproteomics.com
Glycominds http://www.glycominds.com
Contd.
GlycoSuite DB glycosuite.com
Lab-on-a-chip lab-on-a-chip.com
NIGMS Consortium for Functional Glycomics web.mit.edu/glycomics/consortium
Oxford Glycobiology Institute list of publications bioch.ox.ac.uk/glycob/publications.html
Pharmocogenomics Online pharmacogenomicsonline.com
The Brown Lab, Stanford University cmgm.stanford.edu/pbrown
University of New Hampshire Centre for
Structural Biology glycomics.unh.edu
Types of Microarray
Main types of microarrays available commercially are oligonucleotide arrays (GeneChipTM by
Affymetrix) and cDNA arrays (BD Biosciences). Other types of microarray are antibody array,
protein chip array and tissue array. The following text provides a brief description of these arrays.
Oligonucleotide arrays
In oligonucleotide array (or DNA chip), small oligonucleotide (20~80-mer oligos) or peptide nucleic
acid (PNA) probes are synthesized either in situ (on-chip) or by conventional synthesis followed
by on-chip immobilization (http://www.affymetrix.com). Total 11 to 16 copies of a DNA are
spotted for each gene on the array. These DNA fragments have little cross-reactivity with other
genes for minimal non-specific hybridization. Still to combat any non-specific hybridization, a
second probe identical to the first except for a mismatched base at its centre is placed next to the
first. This is termed Perfect Match/Mismatch (PM/MM) probe strategy. To obtain perfect hybrid-
ization, any background hybridization with the MM probe is subtracted from the PM probe signal.
Combination of photolithography and combinatorial chemistry is used for the synthesis of
diverse sequences of probes. Oligonucleotide array allows the simultaneous generation of thou-
sands of probes relatively quickly on the chip surface of 5-inch square of quartz wafer, which is
an ideal substance for adherence of chemicals. This wafer is then washed with a blocking com-
pound, which is then removed by exposure to light. A mask designed with 18-20 micron square
windows is laid on top of array and allows ultraviolet light to pass through areas where a specific
nucleotide is needed. Exposure of light removes the blocking compound from the probe. The wafer
is then washed with a solution of the desired nucleotide that is linked to the same blocking
compound and nucleotide attaches to the probes that were exposed to light, while the nucleotide
attached with blocking compound ensures that all the probes are protected again. A capping step
is added so that probes that did not attach their appropriate nucleotide are not incorrectly synthe-
sized. The process is repeated until all the probes are complete.
In CGH (comparative genomic hybridization), genomic DNA from the patient is labeled with one
fluorescent dye while a normal reference sample is labeled with a different dye, and these samples
are co-hybridized to the array containing the genomic DNA targets. Chromosomal imbalances
across the genome can thus be quantified and positionally defined by analyzing the ratio of the
fluorescence of the two dyes along the targets. The availability of clone sets covering the human
genome opens the possibility for the widespread use of array CGH for both research and diagnostic
purpose. Microarray-based genomic copy-number analysis is now a commonly ordered clinical
genetic test for patients and is defined under various names, i.e., chromosomal microarray
(CMA) and molecular karyotyping. CMA, is used for readily available and easy to handle oligo-
nucleotide arrays originally designed for parallel genomewide analysis of over 10000 SNPs.
cDNA Arrays
The principle of this array is same as oligo arrays, but the probes in this case are larger pieces of
DNA that are complementary to the genes. PCR using specific primers can be used to amplify
specific genes from cDNA to generate the cDNA probes (500~5,000 bases long). A separate PCR
reaction must be performed for each gene. cDNA probes can be mechanically spotted onto a glass
slide. By using this technology one can display 409,000 spots in an area of 1.28 cm2. Hence, all
20,000-25,000 genes of Arabidopsis can be displayed on a single slide. Microarray technique is
highly sensitive as it can detect mRNAs at level of 1/100,000 or 1/500,000.
There are few distinctions between oligonucleotide and cDNA arrays. First, cDNA arrays
eliminate the need for the probe design required for oligonucleotide arrays, while also allowing the
entire genome of an organism to be represented on the array easily, making cDNA arrays more
useful for the analysis of gene expression on a global level. Oligonucleotide arrays have advantage
for their greater hybridization specificity due to PM/MM probe design, resulting in more specific
fluorescence detection.
Protein microarray
It is based on the principle of ligand-binding assay that relies on the formation of product with an
immobilized capture molecule (target) present in the solution. Protein microarrays are now becom-
ing very popular due to their use in the study of antibody-protein, enzyme-protein, DNA-protein
and protein-protein interactions (Figure 12.2). For analysis of protein interactions with other
molecules, protein microarrays have various types of molecules immobilized on the slide surface
Figure 12.2 Protein microarrays: Proteins are immobilized on microscopic slide and the slide
can be probed for various interactions such as identification of antibody, enzymes,
DNA as well as protein. Cy3 is a fluorophore that has been used for labeling of target
such as antibody, enzymes, DNA and protein.
using a contact spotter or a non-contact microarrayer to act as capture molecules. Aldehyde- and
epoxy-derivatized glass surfaces can be used for random attachment of proteins through amines,
and coating the glass surface with nitrocellulose, gel pads, or poly-L-lysine achieves a random
orientation of the proteins as the proteins are passively adsorbed onto the surface (Figure 12.2).
Protein microarrays are of three types, which are described briefly below.
Analytical microarrays
Analytical microarrays are used to measure binding affinities, specificities, and protein expression
levels of the proteins in the mixture. In this technique, a library of antibodies, aptamers, or
affibodies is arrayed on a glass microscope slide and then probed with a protein solution. Antibody
microarrays are the most common analytical microarray. Functional protein arrays are used to
study the biochemical activities of an entire proteome in a single experiment i.e., protein-protein,
protein-DNA, protein-RNA, protein-phospholipid, and protein-small molecule interactions. In re-
verse phase protein microarray (RPA), cells are isolated from various tissues of interest and are
lysed and then arrayed onto a nitrocellulose slide using a contact pin microarrayer. Slides are then
probed with antibodies against the target protein of interest, and the antibodies are detected with
chemiluminescent, fluorescent, or colorimetric assays.
Antibody arrays
Antibody microarray is a powerful chip-based technology composed of hundreds of distinct
monoclonal antibodies printed at high density on a glass microscope slide enabling to monitor the
expression pattern of hundreds of proteins with a single experiment even in a pg/ml range. In this
microarray, glass slides or other chip types with monoclonal antibodies specific against proteins
of interest are attached to their surface. A single antibody microarray can have over 500 antibodies
arrays on their surface. Instead of conducting many Western blot analyses, one can simply use an
antibody microarray to evaluate changes in protein expression levels. This technique does not
measure absolute concentration, but provides a relative measure of protein abundance.
Tissue microarray
To analyze the expression of genes simultaneously in multiple individual tissue samples on one slide,
tissue microarrays are used. The array is composed of 0.6 - 3.0 mm cores of tissue from donor
tissue paraffin blocks, which are arrayed at a high density on a slide. Histochemical and molecular
detection techniques can be used on the slides to allow them to examine gene expression profiling
in disease status across a variety of patients and disease conditions. It is a low cost and high
throughput technique, which could produce material for 500,000 assays and analysed per slide
block with a wide-variety of automated analysis and data collection techniques. The tissue
microarrays allow the entire cohort to be analyzed in one batch on a single slide with identical
reagent concentrations, incubation time, temperature, wash conditions, and antigen retrieval and
can be reused thousand of times with different reagents. The quantitative expression analysis can
be done by H-score system, which is a product of intensity and area stained. The software involved
are TMA-Deconvoluter and Stain finder, Tissue Array Data Analysis, Tissue Array Database (TAD),
TMAJ, etc.
In this technique, 4-5 micrometer tissue sections are produced using a microtome. Specific
areas of interest are selected from paraffin-embedded tissue blocks and are re-embedded into an
arrayed blank recipient blocks. Tissue microarray is of different types i.e., cryo-tissue microarray,
multi-tissue microarray, progression tissue microarray and prognosis tissue microarray.
Glycomics
Glycans are termed the compounds in which sugar residues are covalently attached with the
proteins and biomolecules. An example of carbohydrate microarray is neoglycolipids, spotted onto
nitrocellulose and PVD. The neoglycolipids can be probed with proteins of known carbohydrate
binding specificity to confirm identification of predicted proteinoligosaccharide interactions. This
array technology describes the link of oligosaccharides to the lipid derived from diverse sources,
for example, glycoproteins, proteoglycans, glycolipids, whole cells, organs and synthetic oligosac-
charides.
APPLICATIONS OF MICROARRAY TECHNOLOGY

DNA microarrays are better than other profiling methods (SAGE, SH, PCR) in the sense that they
are easier to use with high-throughput results, and can generate large amount of data in lesser time.
They do not require large scale sequencing and allow quantitation of genes for many samples. Thus
microarray technique seems to have a significant future in diagnostics. The application of DNA
microarray in various fields of research is discussed below in detail.
Changes in Gene Expression Level

With all the techniques to measure gene expression including Northern blotting, differential display,
serial analysis of gene expression and dot-blot analysis, the main problem is that they are unsuitable
for the parallel testing of multiple gene expression. Southerns method of microarray contains
multiple DNA sequences (probes) spotted or synthesized on a relatively small surface allowing
simultaneous monitoring of the expression of thousands of genes, thus providing a functional
aspect to sequence information in a given sample. This technique seems to be ideal for detection
of complex phenotypes and genes whose altered expression underlies complex traits that are
located within genetic regions identified by quantitative traits loci (QTL) techniques. However, SNP
has allowed polymorphisms to be assayed more quickly and also their relevance to disease to be
easily determined. In contrast to the analysis of a single nucleotide polymorphism, gene expression
levels are best analyzed with relatively long probes as in microarray. With long probes, it is possible
to achieve good yields under stringent hybridization conditions. Also the genomic DNA derived
from a normal sample is required for use in the hybridization mixture and whole genome can be
used for expression analysis. To detect mutations or polymorphisms in a gene sequence, i.e.,
change or variation that can occur within a persons DNA sequence, mutation microarray
analysis is used. Microarrays, in combination with defined mutants, can be used to infer signaling
pathways associated with an environmental response i.e., capability to identify common promoter
regulatory elements shared by coregulated genes in cell cycle.
DNA microarrays are also used to examine the gene expression changes under various diseases
i.e., cancer. Tumor profiling using DNA microarray allows the analysis of the development and the
progression of such complex diseases and differential expression in the levels of gene(s) within the
same organism under two different conditions or among two different organisms. Microarrays are
used to identify inheritable markers used in genotyping tool. Thus information about differences
in gene expression between tissue types can help to uncover how our bodies develop sensitivity
and which genes are harmful to target for disease therapy. By the microarray technique, gene
expression studies can also be done for a subset of genes involved in various metabolic pathways.
Microarray expression analysis or expression chip array is used in determining the level, or volume
at which a certain gene is expressed and examine changes in gene expression over a given time
period i.e., within the cell cycle.
Analysis of Sequence Variation

Sequence variation is best analyzed with the shortest oligonucleotides (<15-mer) that will give
specific hybridization to the target site. Multiple genes can be analyzed simultaneously to get a
snapshot of the whole transcriptome of a system at a given time point. Treating mRNA transcript
abundances as quantitative traits and mapping gene expression QTL for these traits has been
pursued in gene-specific ways. Unlike classical quantitative traits, the genetic linkages associated
with transcript abundance permits a more precise look at cellular and biochemical processes.
Microarray provides powerful tools for the genome-wide correlation of gene transcript levels with
physiological responses and alterations in physiological states. It has been applied almost exclu-
sively to a few model species for which the abundant gene sequence data permit the fabrication
of whole-genome microarrays. Microarray technique is also used for mapping human genome
using DNA polymorphisms, which are further analyzed to give enough analytical power to carry
out genetic studies to find the genes associated with common diseases and inherited disease
susceptibilities. It can be successfully applied to nonmodel species to generate new insights of
comparative and evolutionary significance into animal function.
Drug Discovery (Pharmacogenomics)

The goal of pharmacogenomics is to find correlations between therapeutic responses to drugs and
the genetic profiles of patients. Personalized drugs, molecular diagnostics, integration of diagnosis
and therapeutics are the long-term promises of microarray technology. One can examine targets
for drug discovery and potential diagnostic and prognostic biomarkers for many complex diseases.
The use of microarray analysis in drug design and screening allows gene interactions to be studied
with compounds that affect the expression of important genes during drug application. After
identifying a specific molecular target and the development of a series of small molecule inhibitors,
it is necessary to confirm that such inhibitors do indeed act by the desired mechanism of action
on their intended target. In addition, identification of potential on-target and off-target effects, and
pharmacodynamic markers of these effects is highly beneficial for the subsequent preclinical and
clinical studies required for the development of specific drugs.
Toxicological Research (Toxicogenomics)

The goal of toxicogenomics is to find correlations between toxic responses to toxicants and
changes in the genetic profiles of the objects exposed to such toxicants. New drug development
protocols include genomic and proteomic microarray data obtained during preclinical stages of
investigation, but extrapolating this information to humans is not straightforward. However,
microarray data can provide greater insight into and better prediction of the performance charac-
teristics of the product as it moves into clinical phases of development. Recent examples of
microarray in neurotoxicological research combine toxic drug administration with a genetic knock-
out model. Microarray technology has been used to assess toxin induced gene expression abnor-
malities in cancer biology, hepatotoxicity and drug abuse studies. DNA microarray is also an ideal
tool for the identification of bacterial species in a mixed population giving information on both the
abundance and identity of the bacteria in a particular environment.
Diagnosis of Disease
Insights into disease have been one of the most beneficial results of microarray technology.
Expression chips are used in disease diagnosis, e.g., in the identification of new genes involved in
environmentally triggered diseases, i.e., diseases affecting immune, nervous, and pulmonary/res-
piratory systems. For targeted therapies, identification of genes that are either lost during a disease
or typically involved in a cellular function that directly or indirectly prevents the disease from
occurring can be done with a microarray. It is also used to diagnose diseases at very early stages,
so that therapy could be commenced before the disease can cause any harm. Comparative Genomic
Hybridization (CGH) is used to detect any change in the number of copies of a particular gene
involved in a disease state. In this array, each spot of target (large pieces of genomic DNA) in the
array has a known chromosomal location, and hybridization mixture contains fluorescently labeled
genomic DNA taken from both control and diseased tissues.
Molecular karyotyping, can easily and reliably detect unbalanced chromosomal aberrations of
various sizes from as little as 250 ng of DNA on a single microarray, based on fluorescence intensity
information from clusters of SNPs.
Microarray technology has been widely used in studies identifying new genes or molecular
pathways involved in tumor classification, cancer progression and chemotherapy resistance and
sensitivity. It allows the rapid identification of genes that are turned on and off in tumor develop-
ment. Recently a strategy is proposed called SAM (Significance Analysis of Microarrays), which
allows the determination of significantly differentially expressed genes between groups of samples
analyzed by expression arrays i.e., in early and late stages of cancer.
Microarray technology can be a powerful tool to identify unexpected molecular mediators using
animal models that might help in the development of novel targets for improved treatment in
Idiopathic Pulmonary Fibrosis (IPF) and asthma. This can provide a large scale of differentially
expressed genes, which led researchers to shed further light into transcriptional programs involved
in cytokine signaling and apoptosis regulating emphysema, chronic obstructive pulmonary disease
(COPD) and pulmonary fibrosis. Furthermore, this technology is already being applied in respira-
tory clinical pharmacology of complex diseases such as asthma and COPD revealing modern
approaches in therapeutic interventions. Finally, it provided scientists with useful information to

clarify physiological mechanisms underlying the actions of numerous drugs, elucidate the patho-
physiological processes of complex diseases such as IPF, asthma, COPD, lung fibrosis in acute
lung injury and pulmonary edema.
With the help of microarray, one can detect viruses and other pathogens from blood samples
and thus it can be used as a pathogen detection method. Tissue microarray technology is used for
blood cell analysis of patients suffering from red cell disorder and microdissected discs can be used
for PCR-based analysis.
Comparative and Evolutionary Biology

Microarray technology is perfectly suited for sub-classifying otherwise indistinguishable diseases
using straightforward hierarchical clustering techniques and therefore well suited to situations
where just a few genes underpin the condition. This can display pronounced changes in expression
related to an imposed change in physiological status or in response to upstream events in a
transduction pathway. Microarrays have also been used to identify genes that contribute to en-
hanced fitness and assess the changes in gene expression. With the help of microarray, transcript
screening can be done to find molecular basis of natural variation and genotype to phenotype
interaction on an evolutionary scale. Whole genome microarray points toward the environmental
responses of organisms that can be addressed on a global scale with differential expression of a
common set of genes. Transcript expression data across and between the species shows that
closely related species of the same genus may share the expression of many transcripts, whereas
distantly related species will have more divergent profiles. Common gene ontology has been
developed to provide order in a fragmented functional nomenclature by creating a single listing of
attributes to describe objectively gene products in any organism.
Proteomics
Microarray technology can be used efficiently to identify, quantify, and study protein functions
from a global perspective. Protein microarrays are suitable for studying proteinprotein interac-
tions, enzymesubstrate interactions, proteinDNA interactions, protein-lipid interactions, protein
oligosaccharide interactions, proteindrug interactions, protein-receptor interactions and antigen-
antibody interactions simultaneously. Protein microarray is also used in the investigation of kinase
activities, serum profiling, neurodegenerative disorders, correlation of cell phenotype with surface
markers, identification of chromatin-related proteins as well as mapping of WW domains. This
technique is capable of detecting up to 10,000 proteins in parallel. Peptide microarray technology
is used for proteome analyses to study molecular recognition events and the identification of
biologically active peptides. DNAprotein interaction assays have proven useful in the character-
ization and identification of DNA binding proteins. Glycomics is used to study proteincarbohydrate
interactions in biological processes including normal tissue growth and repair, cellcell adhesion and
inflammation, cell growth, fertilization, viral replications, parasitic infection, tumor-cell motility and
progression. Reverse phase protein microarray allows the determination of the presence of altered
proteins that may be due to the result of disease. Proteome chips have been used to screen human
sera for the presence of autoantibodies or viral specific antibodies.
Analysis of Quantitative Traits

Microarray can also be used to know the response of plants or animals towards traits that are
multigene. A good example is response of plants towards environmental stresses such as high
temperature or salinity. Several recent studies have indicated the usefulness of microarray technol-
ogy to understand the response of plants towards salinity. These data have also resulted in predic-
tion of nodal components of stress response pathways in plants.
Assigning Roles to Novel Genes

The most powerful application of microarray technology in biology is to aid in assigning functions
to unknown genes. This is especially useful in the post-genome sequencing era. Based on hierar-
chical clustering, the roles of unknown genes forming a cluster with genes of known function can
be predicted.
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Chapter · December 2012

Rohit Joshi Ratna Karan

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Keywords: Microarrays, differential gene expression, reverse northern, hybridization, oligonucle-

Table 12.1 Chronological order of microarray development.

Year Brief description of the activity

Rauch and coworkers redefined CGH as molecular karyotyping

CONCEPTS AND PRINCIPLES

BASIC STEPS IN MICROARRAY TECHNOLOGY

Expression profile clustering

and visualization are Cyber-T, SAM, BRB-Array Tools (http://linus.nci.nih.gov/BRB-ArrayTools),

Microarray Data Management

Table 12.2 List of some representative websites related to microarray.

APPLICATIONS OF MICROARRAY TECHNOLOGY

Changes in Gene Expression Level

Analysis of Sequence Variation

Drug Discovery (Pharmacogenomics)

Toxicological Research (Toxicogenomics)

approaches in therapeutic interventions. Finally, it provided scientists with useful information to

Comparative and Evolutionary Biology

Analysis of Quantitative Traits

Assigning Roles to Novel Genes

104. Schena M. (2003) Microarray Analysis. Hoboken, NJ: Wiley-Liss. p. 630.

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