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Genechip technology and its applications

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199 Proc. Pakistan Acad. Sci. 42(3):199-204.2005
Muhammad Irfan et al.

Review

GENE-CHIP TECHNOLOGY AND ITS APPLICATION

Muhammad Irfan1, Tayyab Husnain*1, Penny J. Tricker2, Gail Taylor2 and S. Riazuddin1

1
National Centre of Excellence in Molecular Biology, 87-W Canal Bank Road, Lahore-53700, Pakistan, and
School of Biological Sciences, University of Southampton, Bassett Crescent East, SO16 7PX, UK
2

Received April 2005, accepted June 2005

Summary: Gene Chips are finding extensive use in animal and plant science. Generally microarrays are
of two kind, cDNA or oligonucleotide. cDNA microarrays were developed at Stanford University, whereas
oligonucleotide were developed by Affymetrix. The construction of cDNA or oligonucleotide on a glass
slide helps to compare the gene expression level of treated and control samples by labeling mRNA with
green (Cy3) and red (Cy5) dyes. The hybridized gene chip emit fluorescence whose intensity and colour
can be measured. RNA labeling can be done directly or indirectly. Indirect method involves amino allyle
modified dUTP instead of pre-labelled nucleotide. Hybridization of gene chip generally occurs in a
minimum volume possible and to ensure the hetroduplex formation, a ten fold more DNA is spotted on
slide than in the solutions. A confocal or semi confocal laser technologies coupled with CCD camera are
used for image acquisition. For standardization, house keeping genes are used or cDNA are spotted in
gene chip that are not present in treated or control samples. Moreover, statistical analysis (image
analysis) and cluster analysis softwares have been developed by Stanford University. The gene-chip
technology has many applications like expression analysis, gene expression signatures (molecular
phenotypes) and promoter regulatory element co-expression.

Gene-chip occasionally radioactive) tagged nucleotides and

The study of gene expression based on
hybridization of mRNA to high density arrays of
immobilized target sequences that may correspond
to specific genes is called gene-chip technology.
There are two kinds of gene chips, cDNA and
oligonucleotide. cDNA gene chips were developed
by groups led by Patrick Brown and Ronald Davis
at Stanford University [1,2]. The system is based
on standard reverse transcription and PCR
amplification followed by cloning of cDNA for each
gene of interest. cDNA clones are then generally
‘printed’ on glass slides [3] using a robotic printer.
Printing technologies have been developed that allow
printing of 100,000 clones/spots on a single slide.
This system allows for customized arrays [4]. mRNA
obtained from biological samples is reverse
transcribed and end-labelled using fluorescent (or
–––––––––––––––––––––––
Address for Correspondence: Dr. Tayyab Husnain
E-mail: tayyabhusnain@yahoo.com
hybridized to the array. The fluorescence or
radioactive emission from each ‘spot’ is proportional
to the relative specific transcript abundance in the
pool of cDNA hybridized to a cDNA spot.
Abundance of a specific transcript in a sample is
compared to the abundance of that transcript in a
control sample. This technique, although laborious,
delivers a high-density, completely customized system
with high detection specificity.
Oligonucleotide gene chips were developed by
Fodor in 1991 [5] and Lipshutz et al. [6]. In this
system, oligonucleotide probes are directly
synthesized on a solid surface using chemical
synthesis supported by a lithophotographic technique
(http://www.affymetrix.com). Originally, this strategy
was developed to detect DNA single nucleotide
polymorphisms (SNPs) [6]. Later, it was applied to
measure mRNA expression. In Affymetrix
Arabidopsis arrays, each gene is represented by
Gene-chip technology and its application
20 sense 25-base-long oligonucleotide probes and,
as a control, by 20 mis-sense probes that have one
base mismatch in the central part of the probe--
usually a base is substituted with a complementary
one. Probe sequences are obtained from publicly
accessed databases and, in most cases, cover the 3'
parts of the coding sequence to increase the sensitivity
of the assay and correct for SNPs. RNA obtained
from biological samples is processed prior to
hybridization. Double-stranded cDNA is obtained
from total or mRNA (DNase treated) using reverse
transcription. cRNA is then obtained using in vitro
transcription with biotinylated nucleotides, and after
fragmentation is hybridized to an oligonucleotide
array. If biotinylated nucleotides are used to
synthesize cRNA, the array is visualized by staining
with streptavidin-phycoerythrin conjugate. The value
representing expression of each gene is calculated
based on fluorescence derived from each sense
probe as compared to emission from each mis-sense
probe. If the difference between fluorescence emitted
from a set of sense probes and mis-sense probes is
zero, the particular gene is not expressed in the
sample.
Construction of a Gene-chip
Preparation of the sample
Gene chips may be used to measure gene
expression levels in different ways. One of the most
popular gene chip applications is to compare the
gene expression level in two different samples, e.g.
the same cells or cell type under two different
conditions. This is based on labeling mRNA
extracted from one condition with one dye, e.g.
green, and from another condition with a different
colored dye, e.g. red. The hybridized gene chip is
excited by a laser and scanned at wavelengths
suitable for the detection of the red and green dyes
as shown in Figure 1. The amount of fluorescence
emitted upon laser excitation corresponds to the
amount of nucleic acid bound to each spot. If the
200
nucleic acid from the sample in condition 1 is in
abundance, the spot will be green, while if the nucleic
acid from the sample in condition 2 is in abundance,
it will be red. If both are equal, the spot will be yellow.
If neither is present, it will not fluoresce and so appear
black. Thus, from the fluorescence intensities and
color for each spot, the relative expression levels
for the genes in both samples can be estimated. In
this way thousands of data points, individually
providing information about expression of a particular
transcript, can be obtained from a single experiment.
Figure 1. Microarray: General Functional Principle.
The complexity of gene chip analysis means
that tissue sample collection becomes a crucial factor
in the data produced. As gene chips have been used
with increasing frequency in recent years, the amount
of diversity in gene expression between samples,
even from the same tissue in the same individual, has
become clear. Precise sampling (including factors
such as the time of the day and month taken) and
the ability to sample homogeneous tissues, by using
techniques such as laser capture microdissection [7]
are crucial steps in obtaining accurate analysis. At
the same time, the amount of tissue required can be
a problem as a relatively large amount of RNA is
required. However, more recent techniques to
amplify RNA have been developed that allow
extraction from minute tissue samples [8]. Brown’s
group [9] at Stanford University has described a
pool of RNAs derived from 11 diverse human tumor
201
cell lines that has become a kind of de facto universal
human reference RNA. Recently a labelled
oligonucleotide that is complementary to every
feature on an array has been shown to be an effective
reference, without the complication associated with
references derived from mRNA [9]. Ultimately, the
development of gene chip technology or other
processes to allow high-throughput parallel measures
of absolute RNA abundance are needed to provide
a robust description of the transcriptome of specific
cellular lineage, development stages and disease
states.
RNA Labelling protocols
Expression analysis labelling protocols are
based on the reverse transcription of mRNA, either
from highly purified poly (A) mRNA or total RNA
extract. Extensive purification of RNA is essential to
remove all contaminating proteins, polysaccharides
and other organic material, especially RNases. Many
protocols have been developed for the extraction of
high-quality RNA using various in-house and
commercial kits and reagents. Initial protocols for
target labelling, whereby reverse transcription of
mRNA is primed, used a poly (dT) primer in the
presence of fluorescently labelled nucleotides
(typically cy3- or cy5- conjugated dCTP or dUTP).
Cy3- or cy5-conjugated nucleotides are bulky,
however, which makes their incorporation using
standard enzymes very inefficient. In addition the rate
of incorporation can differ between dyes, potentially
resulting in dye biases.
In an alternative method, an amino allyl
modified dUTP is used instead of pre-labelled
nucleotide. After reverse transcription, the free amine
group on the amino allyl modified dUTP can be
coupled to a reactive N-hydroxysuccinimydl ester
fluorescent dye. Although this technique is longer than
direct labelling, its benefits, including better sensitivity,
fewer dye biases and decreased costs, seem to be
worth the extra effort (http://www.microarrays.org/
Muhammad Irfan et al.
protocols.html).
Hybridization
The incubation of the target with the gene chip
is typically performed at 65 0C for aqueous
hybridization and 42 0C if 50 %( V/V) formamide is
present in the hybridization solution, in a manner
similar to protocols used during other hybridization-
based techniques. The hybridization of gene chips
generally occurs in the minimum volume possible.
Many laboratories have adopted special chambers
to enclose gene chips to enable their immersion in a
waterbath to keep them at a constant temperature
during the hybridization step. The hetroduplex
formation in the hybridization reaction is largely
determined by the concentration of DNA on the glass
slide. To ensure the hetroduplex formation, 10-fold
more DNA is spotted on the slide than in the solution.
Other factors that can influence the rates of
hybridization include the presence or absence of
monovalent cations and/or formamide, the
hybridization time, temperature and the length and
specific activity of the probes. Following
hybridization, the gene chip is washed repeatedly to
remove the unbound and non-specific signal and is
then ready for the acquisition of the image. The
hybridization kinetics of the two labelled probes are
different, Wang et al., [10] have developed a
method that makes this validation easy to implement
to ensure reliability in two-color gene chips.
Image acquisition
The readout from the gene chip is captured as
an image that is acquired using a scanner for
fluorescent signal detection or a phosphorimager for
the detection of radioactive signals. The scanners
currently available use either scanning confocal laser
technologies or a charge-coupled device (CCD)
camera. Most current scanners are designed to
detect four wavelengths of light emission, but a
scanner with the capacity to read up to five different
Gene-chip technology and its application
wavelengths on any one slide has recently been
released.
Standardization
Gene chips are affected by the differential
labelling and hybridization efficiencies of the targets.
Traditional housekeeping genes or maintenance genes
can be included on all arrays to assist in normalizing
individual hybridization differences. Another
approach is to spike each sample with a cDNA that
is present on the array but not present with the control
or test sample to act as an internal control for the
labelling and hybridization processes [11].
Given the multitude of platforms and
methodologies, a growing concern for researchers
using gene chip information is a reporting standard
for all gene chip experiments. This allows researchers
to assess all aspects of experimental design and
execution of array experiments. This led to the
creation of a Minimum Standard About Gene Chip
Experiment (MIAME) compliance. Major publishing
houses now advocate MIAME compliance with any
published DNA gene chip experiments [12].
Information regarding the requirements for MIAME
compliance, design of experiments and data storage
can be found at the Gene chip Gene Expression Data
Society website (http://www.mged.org/
Workgroups/MIAME/miame.html). The European
Bioinformatics Institute (EBI) has also created a
public database where MIAME compliant data can
be stored and uploaded in XML data exchange
format Gene Expression Markup Language
(MAGE-ML., http://www.ebi.ac.uk/arrayexpress/
[12] and Boussioutas and Haviv [13]). Once
obtained, raw DNA array data must be processed
before biologically relevant information can be
extracted. This is one of the most challenging aspects
of transcriptome analysis. There are many sources
of variation in gene chip experiments. These include
the amount of DNA delivered to each individual spot
on the array, which in turn can be affected by PCR
202
efficiency, pin geometry, and the degree of DNA
fixation to the array surface. In addition, the quality
of starting RNA, reverse transcription, and labelling
efficiency can vary, as can the hybridization efficiency,
amount of non-specific hybridization, amount of
overlap from neighboring spots and image analysis
[11]. Using at least three replicates in an experiment
reduces the number of false positives and false
negatives providing more reliable results overall [14].
In addition, normalization methods, usually using a
set of housekeeping genes or other control genes,
can be applied in an attempt to account for this
inherent variability [15,16].
Analysis
Although gene chip technology is still being
refined, the basic methods of analysis are well
established. An image analysis program, in most
cases, is used to calculate the quantitative ratio of
the level of gene expression between the two samples
being compared. Before the software is able to
interpret the intensity value for each probe a grid
must be overlaid on the image of the gene chip to
map the location of pixels representing each spot. A
software package for initial image analysis was
developed at Stanford University (http://
www.rana.stanford.edu/software/) and is freely
available for academic users. Most scanners are sold
with an option of an image analysis software package
such as Imagene (Biodiscovery, Los Angeles, CA,
USA) Quantarray (GSI Lumonics) or GenePix 2.0
(Axon instruments, Foster City, CA, USA). Some
of these packages use cluster analysis algorithms to
group the genes on an array according to their
expression profiles over a number of experiments.
A version of this type of program, called Cluster, is
also available from Stanford University.
Bilban et al. [17] introduced a method to filter
false-positives and false-negatives from DNA gene
chip experiments. This was achieved by evaluating
a set of positive and negative controls by receiver
operating characteristic (ROC) analysis. An
203
advantage of this approach is that users may define
thresholds on the basis of sensitivity and specificity
considerations. The area under the ROC curve
allows quality control of gene chip hybridizations.
This method has been applied to custom-made gene
chips developed for the analysis of invasive
melanoma derived tumour cells and yielded fewer
mis-classified genes. Provided that a set of
appropriate positive and negative controls is included
on the gene chip, ROC analysis obviates the inherent
problem of arbitrarily selecting threshold levels in
gene chip experiments. The proposed method is
applicable to both custom-made and commercially
available cDNA gene chips and can help to improve
the reliability of predictions from gene chip
experiments.
Applications
Clustering the expression data can identify
groups of genes with similar expression patterns that
may be controlled by shared regulators. In carefully
designed time course studies, gene chips also allow
the temporal sequence of transcription induction or
repression to be followed. Such information is useful
for determining the order of events during the
activation of plant defense responses and separation
of primary and secondary effects triggered by an
infectious agent or signaling molecules. The large-
scale nature of the survey using gene chips also
produces a ‘gene expression signature’ that is a new
form of molecular phenotype. Gene chips can also
be used to find the similarities or differences between
different treatments (e.g. the effect of pathogen
infection or chemical signals or hormones on gene
expression) and to determine the level of potential
cross-talk between different the signaling pathways.
Another novel use of gene chips is the direct
identification of DNA binding proteins [18]. Thijs et
al. [19] modified an original Gibbs sampling
algorithm for motif finding [20] and looked for
potential promoter regulatory elements in the
upstream region of co-expressed genes.
Muhammad Irfan et al.
Acknowledgements
Financial support of Commonwealth
Scholarship Commission, United Kingdom, to
Tayyab Husnain (Reference No. PKCF-2003-218)
and from Higher Education Commission, Pakistan,
to Muhammad Irfan (PIN 041201588 B-027) is
gratefully acknowledged.
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