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Microarray Technology
 Description of Module

Subject Name

Paper Name

Module Name/Title Microarray Technology

Dr. Vijaya Khader

Dr. MC Varadaraj

Microarray Technology
1. Objectives

 Comprehend how Microarray has developed as a technology for gene level studies

 Explain the different methods of array production and technical aspects

 Explain the process of data acquisition and biological interpretation in microarray

 Demarcate applications of DNA microarray and advancements in genotyping, gene

expression analysis

2. Concept Map

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 3. Description
3.1. Prologue to the technology

Microarray technologies infer the combinatorial interface between a set of biomolecules

(DNA fragments and proteins or their parts thereof), with encoded library of molecular
targets immobilized on a substrate that could be glass slide or silicon chip.
The literal meaning of a DNA array is a designated arrangement given to the molecular
targets which could be unique sequences individually synthesized on the solid planar surface
or pre-synthesized probes (oligonucleotides or PCR products) attached to the array platform.

3.2 Introduction

Microarray analysis is a revolutionized biological information processing technique as it

offers a platform for analyzing large number of nucleic acids, peptides, proteins, cells on a
single substrate in parallel at one particular time. It’s a multidisciplinary technique that
followed advancements in different disciplines and techniques, molecular biology
hybridization methods, fluorescence imaging, analyses using diagnosis of sensitivity results.
The convergence of ideas and working methodologies in molecular biology has enthused the
thrust provided by the technology advancements in preparing miniaturized collections of
biomolecules (DNA, cDNA or proteins) on solid substrates, have all appended to the
evolving field of microarray and microchip technologies.
Microarrays are electronic platforms that are made using microelectrodes, providing
characteristic indications of sensitivity, specificity, momentum, accuracy and multiplexing for
genomic and proteomic applications that facilitate robotic and controlled analyte

Microarray Technology
concentration, DNA hybridization, stringency and parallelism. The principle features of
microchips include the following criterion:
i. Miniaturization
ii. Parallelism
iii. Multiplexing
iv. Hybridization sensitivity, specificity
v. Oligonucleotide addressing
vi. Automation
vii. Statistical analysis
viii. Bioinformatics
ix. Nanotechnology

The amount of quantitative data generated using the microchip platform is a major challenge
for biological experimentation and analysis. Bioinformatics resources amalgamating
information processing, mathematical models and statistical analysis have generated the
much required biological analysis from the microarray chips.

3.3 Principles of Microarray

Exploring the hybridization of nucleic acids is a central molecular biology feature that
transverse from conventional gene expression analysis approaches such as Northern blotting,
Reverse transcriptase polymerase chain reaction (RT – PCR), nuclease protection assays to
high throughput analytical scales by the use of microchips. DNA microarrays have provided
a formidable tool that outperforms the traditional techniques in terms of not just numbers but
also sensitivity as well as specificity. We sail through a journey to understand the microarray
technology, by selecting fabrication of production of the chips to experimental designs and
analysis. The underlying idea of DNA microarray technology is to immobilize known DNA
oligos or sequences referred to as targets / probes (interchangeable terminology used by the
scientists) in micrometer sized droplets on a solid substrate (microchip) and explicitly

Microarray Technology
hybridizing complementary sequence of the analyte DNA or a probe / target. Fluorescently
labelled reporter systems identify or quantify the presence or absence of particular targets
aka genes in the hybridization extract. So, essentially following the principle of northern
blotting for gene expression analysis, manoeuvring filter based immobilizations to combining
fluorescence with microscopy and automation of oligonucleotide deposition, macroarrays
have now been re-synthesized as miniaturized microarrays.

3.4 Nomenclature of Microarray Analysis

Scientists undertaking microarray experimentation have used interchangeable definitions for

‘probes’ and ‘targets’. Here, we denote the key components of all microarray systems that
would be designated as follows:
 Array Substrate – solid support that immobilizes nucleic acid sequences, or ‘targets’
 Probes – one or more labelled samples (hybridization extract), that are complementary
to targets and hybridized with microarrays
 Targets / Spot – Nucleic acid sequences immobilized on microchip / experiment or
feature that is identified after hybridization
 Hybridization signal detection and quantification system

3.5 Microarray types

DNA or Oligonucleotide Microarray
cDNA or Gene expression microarray
Protein Microarray
Tissue Microarray

3.6 Fabrication of Microarrays

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3.6.1 Probe Labeling
The microarray sample being analyzed in the experiment, be it mRNA or aRNA or cDNA
tested for gene expression analysis or DNA derived from genomic analysis, is converted to a
known labelled population of nucleic acids, the probes. The probes uniquely characterized as
numerous differentially labelled nucleic acid fragments numbered in thousands per
microarray slide. The range of the microarray probes immobilized in terms of their number
as well as their sequence variability dictates that the routine molecular biology
experimentation has structured here manifold, hence increasing the complexity and output of
the microarray slides. This demands highly sensitive methods of immobilization of the
probes as well as stringency in terms of hybridization experiment, which annuls background
spotting of hybridization on the microchip.
Cyanine dyes Cy3 and Cy5, have been scrutinized to be utilized for labelling of the probes,
due to their discrete absorption and emission spectrum. The use of fluorescent labels has the
added benefit of permitting the distinctive detection of two or more signals during
experimentation, so as to look for comparative analytical platforms with increased accuracy
and throughput of microchip analysis.

3.6.2 Genetic content of microchips - Technology platforms

Microarrays composed of different types of genetic content or technology platforms utilized

in molecular level characterization whose identity determines the class of quantification
aimed as in cDNA Arrays (purified or amplified PCR products of cDNA clones),
Oligonucleotide arrays (pre-synthesized oligos and in situ synthesized oligos). Quantification
of multiples of tens or hundreds to thousands of targets with one or more labelled probe is
the level of interrogations that are applied using the DNA chips here, that are pitched to yield
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specific and accurate information by hybridization. Several strategies for preparation of the
immobilized targets or nucleic acids exacting the oligonucleotide addressing are in practice to
generate varied technology platforms with deposited DNA fragments as described below.
i. Printed Oligonucleotides arrays
ii. cDNA arrays

Printed oligonucleotide arrays – Oligonucleotides, which are short stretches of DNA

fragments can be attached or deposited as intact or modified DNA on specially treated array
surfaces. The mechanism of microchip fabrication is described further (--). The length of
oligos typically varies between 30-70 nucleotides, where intended sequences of such targets
can be customized according to the researcher’s need. Oligonucleotide arrays have several
advantages over cDNA arrays listed as:
 Same gene with multiple targets can be designed to sufficient probe quantification in
the same experiment
 Oligonucleotides offer a tight control over the genetic content on the arrays, so as to
demarcate alternate splicing variations, alleles, related gene sequences with a judicious
choice of oligos attached
 Time, resources and efforts to prepare cloned targets used in cDNA makes
oligonucleotide printing best commercially suited chips

Design of oligonucleotides for arraying – The design of oligonucleotide targets cater to

influence the specificity and strength of hybridization analysis which make use of
computational algorithms to benefit the researcher have advanced options of known target
sequences. The general rules for selecting target oligonucleotides (oligos) have been carefully
enlisted after experimentation as:
a) Repetitive genome elements, palindromes and repeat polynucleotide content are
avoided
b) Specificity is maintained be careful selection of short homologous regions to avoid
cross-hybridization between genes

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 c) Eventual even distribution of all the nucleotides is desirable, with length and
mismatch positions influencing the overall hybridization efficiency

cDNA arrays – cDNA clone libraries, expressed sequence tags (ESTs), exome sequences,
clones from subtraction libraries, PCR-amplified open reading frames are the targets that are
mostly applied in cDNA platform microchips. The development of mechanical microspotting
methods and instruments have revolutionized the cDNA chips which are contended with a
big role as gene expression microarrays customized according to different experimental
needs.
Preparation and desired properties of DNA targets:
a) PCR amplification of targets from clone libraries, or RNA of tissue, or bacterial
cultures
b) Optimal length of DNA targets is between 200-800 nucleotides, which form specific
and stable hybrids on microchips with UV immobilized DNA targets on aminosilane
treated chips for increased specificity

Non-homologous DNA fragments or single stranded DNA generated by asymmetric PCR or

by exonuclease digestions as well as excess amounts of target DNA are needed to increase
specificity of microchip analysis.
3.6.3 Control targets

The multiplexed and parallel arraying process of the number of targets on the microchip
necessitates the need of effective controls during hybridization as well as preparation of
microchips. The standardization of the array deposition as well as experimentation is
addressed by such control targets on the microarray platform so as to accommodate the
variability generated due to labelling efficiency, printing or depositions of targets,
hybridization conditions and slide surface chemistry as well as spatio-temporal variations on
the chip. The validation of microchip results can be proceeded optimally use of the tabulated
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control targets. The control elements serve as quality indicators and cover for deviations in
labelling of targets between dyes, slide printing as well as hybridization issues.

Table 1: Description of the controls in the microarray experimentation

Control Control Structure Function
Category
Spike - In Exogenous known sequences with Monitoring of microarray
no eventual expression alteration processes
under experimental conditions
Blank No DNA sequences Maintaining hybridization
stringency
Positive Pooled Genomic DNA Labelling and hybridization
strictures
Negative Unrelated DNA pool Hybridization specificity and
detection limits
Ratio Control Differential controlled amounts for Labelling efficiency check, colour
each dye spiked in as gene discrimination and hybridization
sequences before labelling check
Dynamic Range Molar level controls of sequences Labelling range check,
Control spiked in before labelling hybridization and saturation
checkpoints

3.7 Fabrication and array manufacturing

Microarray analysis is a high-throughput miniaturized hybridization technique which uses
fluorescently labelled targets and probes in large numbers to generate vast data for biological
analysis. The fabrication of microchips depends upon the technology platform being
addressed i.e. would depend upon the query and hypothesis behind the analysis, so as to
lead us towards understanding experimental design, layout of arrays and production details.
The following section deals in understanding the basics of all these factors in such a
technology setting.

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3.7.1 Microarray manufacture
1. Microarray slide surface chemistry – generation of target specific linkage on chips
2. Deposition of targets – pre-synthesized versus in situ oligo deposition
3. Probe labelling methods – direct, indirect and random priming

Microarray slides have been developed mostly by two parallel approaches i.e. direct
synthesis of nucleic acid targets onto the microchip, or purified targets pre-synthesized onto
the solid surface which are capacitated for binding the targets. Variations on the original
method of array production have been followed by various scientists, though the basic
strategy of binding remains as surface modification of the slide according to the charge
distribution to enhance ionic interactions of the target onto the surface, tethering of the target
using surface chemistries, washing away of unbound DNA fragments.
a. Pre-synthesized arrays – The technique follows the classical method developed by Ed
Southern and followed intensively by Patrick O. Brown as the robotic spotting of small
volumes (nanolitre or picolitre range) of DNA targets onto poly-lysine or poly-amine
coated slides utilizing their potential of electrostatic adsorption. This has led to in
house manufacture of gene chips according to the laboratory requirements and
customized glass microarrays were prepared using this where 400 to more than 10,000
spots can be administered to each slide using contact based or non-contact deposition
pins. The suitable attachment chemistries include oligonucleotides which are treated
with amines to bind aldehyde slides, imidazole derivatization of 5’ phosphate groups
followed by tethering to amine solid surface, and bifunctional cross-linkers coupling
the aminated oligos to amine derivative slides to name a few methods of tethering
DNA fragments resulting in the covalent linkage between the slide and amplicons. The
cDNA microchip is usually prepared using the deposition methods from cloned cDNA
libraries, with a general theme of labelling the target using reverse transcription of
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 RNA using labelled deoxyribonucleotide triphosphates to produce cDNA as double-
stranded target on the chip.
Printed oligonucleotide arrays are prepared using analogous phosphoramidite
chemistry that allows reactive group addition for covalent coupling to the array
surface producing homogenous sequence and spot size uniformities with upto 50,000
features per array that are 80-200 μm in diameter. The probe sequences featuring on
the oligonucleotide arrays are predictably deliberated to induce optimal target
specificity and uniformity distribution of hybridization execution. The length of the
probe, unique significant sequence selection with limited homology with non-relevant
transcripts in the genome, uniform length, density and composition of nucleotide
bases are maintained to have specificity stringency of chip hybridization. Spots or
characteristic features on the array therefore represents distinct targets which range
from 25-80 bases without compromising specificity and sensitivity of the technology.
Companies like Agilent have commercialized single stranded nucleotide microarrays
in the sense orientation used for antisense cDNA or aRNA (amplified RNA)
hybridization.

b. In-situ arrays –In situ probe synthesis was pioneered as ‘on chip’ synthesis by Fodor et
al. and commercially available as Affymetrix GeneChipsTM as a popular chip platform
for gene expression profiling studies. The process involves computer aided algorithm
based design of photolithographic masks that ensure nucleotide binding as per the
requisite details given by the set of oligonucleotide probes that serve as a base of
complementation. The glass chip is photo-protected and illuminated through physical
holes in the photolithographic mask at selected spots on the slide. The photo-

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 protective masked nucleotides are allowed to couple where the photons of light had
illuminated the addressed site and the process repeated to generate distinctive targets
on the slide of defined length and sequence. An example of the process is highlighted
in the given figure. Several variations on the this theme have been made available in
the market by different companies as also Nano-chip manufacturing gets more
popular with time, where uniformity of target set and ink jet printing is routinely
employed to get in situ manufactured chips and standard phosphoramidite chemistry
is employed for chip manufacture.

Figure 2: Oligo synthesis as targets on microchip proceeds as step wise addition of light protective
nucleotides, which are masked. Photolithography proceeds when a physical mask is illuminated
at selected sites to let the selective appendage of nucleotides so as to generate distinctive
sequences at each site so as to generate anchored targets.

3.8 Microarray Hybridization Technique

Hybridization in biological analyses is undertaken for identification and quantification of
analytes within larger sample datasets. Complementation annealing of single stranded
nucleic acids to target strands is the well established biological identification technique and
this is the sound basis of binding of probe molecules to the targets on the chip substrate. The
critical analytical factors that are a part of the technique are summed below.

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  Pre-hybridization blocking – for effective target attachment and blocking of nonspecific probe
binding
 Hybridization stringency – use of automated instruments in controlled environment and
hybridization chambers
 Hybridization buffering – for ensuring stable pH and kinetic enhancement of hybridization
 Stringency washes – check for low homology complementation between target and probe and
less background noise

The hybridization of the target tethered to the chip and the probe practically are expected to
form duplexes as complementation is involved. The targets are treated with the hybridization
extract from the samples to be analyzed and form duplexes. The labelled targets and probes
then are scanned by the scanning fluorimeter to generate hybridization data as relative
binding patterns which directly represents the amount of genes which are expressed in the
sample probed with the target oligonucleotides. The comparative duplex formation during
hybridization predicts the relative trends of probe and target specificity and kinetics of the
reaction. Multichannel arrays present the possibilities of using two or more labeled samples
(different fluorescent dyes) with simultaneous hybridization at the chip. The relative
abundance of each sample is measured by their target specific associations and intensity
differences.
Steps of Microarray Experiments
Irrespective of the specific technology employed, a microarray experiment consists of three
basic steps: sample preparation and labeling, sample hybridization and washing, and
microarray image scanning and processing. In the following section, the cDNA microarray
will serve as a basis for a general discussion of these steps. In general, experiments based on
other platforms, such as the Affymetrix GeneChip, follow similar principles unless
specifically stated. The general schema of a cDNA microarray is illustrated in Figure.

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Fig. 3.3 cDNA microarray schema. Templates for genes of interest are obtained and amplified by PCR, printed using high
speed robot. Total RNA from both the test and reference sample is fluorescently labeled with either Cye3- or Cye5-dUTP
(Deoxyuridine Triphosphate) using a single round of reverse transcription. The fluorescent targets are pooled and allowed to
hybridize under stringent conditions to the clones on the array. Laser excitation of the incorporated targets yields an
emission with a characteristic spectra, which is measured using a scanning confocal laser microscope. Monochrome images
from the scanner are imported into software in which the images are pseudo-colored and merged. Information about the
clones, including gene name, clone identifier, intensity values, intensity ratios, normalization constant and confidence
intervals, is attached to each target. Data from a single hybridization experiment is viewed as a normalized ratio (that is,
Cye3/Cye5) in which significant deviations from 1 (no change) are indicative of increased (> 1) or decreased (< 1) levels of
gene expression relative to the reference sample. In addition, data from multiple experiments can be examined using any
number of data mining tools.

3.9 Image Scanning, Pre-processing and Data Analysis

Standardization and Normalization of microchips

Microarray scanners in general contain two different laser wavelength cut-offs that have
representative absorption and emission spectrum. The fluorescent dyes use in most of
microchip analysis are Cy3 and Cy5, so a green and red scan of the laser beam gives two
image analysis, which are super-imposed to generate a pseudo-color final images. The
images are derived using a confocal microscope based detector system and the use of
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samples which are replicated and the characteristic controls at the chip finally generates
enormous amount of data set points which are also referred to as features. The high
resolution images are then pre-processed to gauge the quality of the labeling, hybridization
and fluorescence detection before they can be assessed for result analysis. This process is
termed as pre-processing of the images as a part of the normalization, so as to ascertain the
high-throughput management of the data obtained. To gain maximum insights from the chip
experimentation, data processing using specialized statistical parameters and software’s is
done to normalize the data thereby removing experimental variations, and finally data
transformations to derive biologically relevant conclusions from the chip hybridization.
Array Scanning and Data Analysis

Microarray analysis involved the following procedures to get to biologically significant

quantifications.

Microarray Data Cleaning - Image pre-processing

The pseudo-color image formulated by the fluorescent intensities of the features is converted
from a TIFF format into numerical values that can be quantified as gene expression analysis.
The background signal is subtracted to attain the original spot intensity. The steps that are
followed for image processing are enlisted below.
The image is viewed as gridded pattern of spots and processed further as per the scheme
described
1) Spot Identification – indexing individual spots in image and estimation of their sizes
2) Image Segmentation – moulding the gridded areas to locate good, bad and flagged
features
3) Spot Quantification – calculating the intensity of each feature after noise cancellation
and asserting them numerical values
4) Spot Quality Assessment – assessing the entire array for data reliability and
experimentation control

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Data Normalization and transformation
The processed images are then allocated for final data analysis using normalization steps to
transform data to executable biological interpretations. Normalization practically removes
systematic variations that could arise from concentration of DNA printed on the chips, print-
tip variations, fluorescence labelling efficiency and bias, spatial microarray slide variability,
differential incorporation of the two dyes in the DNA targets / probes used in
experimentation. The best estimations of colour intensities are done by transformations using
various statistical parameters that take care of the bias associated with the multiplexed
experiments on the microchip so as to attain even distribution of experimental features over
an intensity range.

Visualization and Clustering

A series of eventual steps to transform imaging data into numerical values is achieved by
applying metric investigations and then the data is visualized to score for uniformity using
graphical form. The final transformed data is then clustered according to supervised or
unsupervised learning that are dependent upon the sort of results desired from the
experiment, giving a meaningful trend of the microchip experiments that is ensured by the
control and replicate multiple features of microarrays.

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 3.10 Applications of microarray analysis
Microchip analyses as a high throughput formula has found its applications in understanding
complex biological queries broadly clubbed under the following parameters:
 Oligonucleotide arrays as tiling arrays in genomic sequence analyses
 Genotyping arrays as allele quantification in genome wide association studies
 DNA microarrays in SNP analysis using comparative genomic hybridization
 cDNA arrays for gene expression profiling

Microarray provides for as a resourceful tool that is converting time consuming analyses to a
rapid high power analytical platform after the advent of genome level sequence
determinations. The miniaturized and multiplexed target and probe hybridization leads to
generation of thousands of experimental features and combining these data points, global
scoring of biological events and processes can be performed simultaneously.

Genomic sequence analysis

Tiling arrays prove a useful tool for genomic sequence estimations and genomic annotation
as well as for elucidating novel genomic content from open reading frames in the sequence.
Gene regulatory networks can be experimentally addressed by advanced analyses of
transcription factor – gene interactions by performing immunoprecipitation of DNA-
transcription factor complexes on microchips popularly called ChIP on chip.

Genotyping analysis
Oligonucleotide microarrays have utilized to harness genome level single nucleotide
polymorphism and mutation analyses, prediction of splice genetic variants and are grouped
under genomic mismatch scanning and representational difference analysis. The candidate
genes that correspond to such differences are usually implicated in disease gene analysis that
catalogues genotypic variation scores by either SNPs or mutation genetic component
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prevalent amongst ethnic population trends and thus prove handy information for
understanding complex disease patterns. SNP chips can interrogate linkage and linkage
disequilibrium scores amongst individual populations, so that global interpretations can be
made from their hybridization profiles. SNP based molecular diagnostics can be followed
after their detection as they serve now as biomarkers for diseases as well as in forensic
detections.

Gene expression analyses

The aspects of gene expression analysis is studied by various techniques by traditional means
which encompass Northern blotting, Real time RT-PCR, Nuclease protection assays,
differential display, subtractive hybridization, representational differential analysis, serial
analysis of gene expression. The basic paradigm of all such technical intervention rests at
measuring the messenger RNA content or identification of the mRNA transcripts by scoring
them digitally or through their presence or absence at particular dynamic cell conditions.
Microarray platforms offer the opportunity to relate to the transcriptome as well as proteome
profiling of the activity of genes at different environmental and spatio-temporal scales.
Relative abundance or expression criteria of specific transcripts can be investigated to
understand complex gene patterns in biological samples using fluorescent labels and image
quantification of the obtained microchips. Such analyses have been performed widely on
most prokaryotic and eukaryotic systems, for understanding the concepts ranging from
development genetics, expression patterns in model organisms, gene distributions during
diseases and in pathogenic versus non-pathogenic microbes as well as drug treatment
differences. All such analyses have appreciated the formulation of novel drug candidates,
biomarker discovery, comprehensive metabolic networks, disease gene finding, selective
environmental effects, immunomodulations, pathophysiology and epigenetic study designs
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as microarray imprints the organism’s expression sketch, quantifying the level, variability
and differential prototypes as ‘signature’ biology of the complete system.

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