Microarray by Jamal

Name:.
Jamal Habib
Submitted To:. Mam Sidra
Subject:. Adv. In diagnostic technique
Semester:. 8th
Microarray technology
Microarray analysis techniques are used in interpreting the data generated from experiments on
DNA, RNA, and protein microarrays, which allow researchers to investigate the expression state
of a large number of genes - in many cases, an organism's entire genome - in a single experiment.
Microarray Principle
mRNA is an intermediary molecule which carries the genetic information from the cell nucleus to
the cytoplasm for protein synthesis. Whenever some genes are expressed or are in their active
state, many copies of mRNA corresponding to the particular genes are produced by a process
called transcription.
These mRNAs synthesize the corresponding protein by translation. So, indirectly by assessing the
various mRNAs, we can assess the genetic information or the gene expression. This helps in the
understanding of various processes behind every altered genetic expression.
Thus, mRNA acts as a surrogate marker. Since mRNA is degraded easily, it is necessary to convert
it into a more stable cDNA form. Labeling of cDNA is done by fluorochrome dyes Cy3 (green) and
Cy5 (red). The principle behind microarrays is that complementary sequences will bind to each
other.
The unknown DNA molecules are cut into fragments by restriction endonucleases; fluorescent
markers are attached to these DNA fragments. These are then allowed to react with probes of
the DNA chip. Then the target DNA fragments along with complementary sequences bind to the
DNA probes.
A computer is used to record the pattern of fluorescence emission and DNA identification. This
technique of employing DNA chips is very rapid, besides being sensitive and specific for the
identification of several DNA fragments simultaneously.
BASIC STEPS IN MICROARRAY TECHNOLOGY
Steps involved in cDNA based microarray:

. Sample collection.
. Isolation of mRNA.
. Creation of labeled cDNA.
. Hybridization.
. Collection and analysis.
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 labeled with either Cy3 (green) or Cy5 (red) fluorescent dyes.
.The samples are combined and hybridized to the microarray.
. After that washing was done to remove non-hybridized target, laser scanning was carried out
and the emissions measured in each color channel.
. Specialized software is used to measure intensity ratios to each spot, which are then exported
for statistical analysis to identify differentially expressed genes.
Applications
In cancer
Tumor formation involves simultaneous changes in hundreds of cells and variations in genes.
Microarray can be a boon to researchers as it provides a platform for simultaneous testing of a
large set of genetic samples. It helps especially in the identification of single-nucleotide
polymorphisms (SNPs) and mutations, classification of tumors, identification of target genes of
tumor suppressors, identification of cancer biomarkers, identification of genes associated with
chemoresistance, and drug discovery. For example, we can compare the different patterns of
gene expression levels between a group of cancer patients and a group of normal patients and
identify the gene associated with that particular cancer.
Antibiotic treatment
Increase in the number of resistant bacteria and superadded infections has led to failure of
antibiotics. Virulence of the bacterial strains too affects the outcome of the disease process. In
oral cavity where anaerobic bacteria might be the infective agent, they often are not easily
culturable, especially organisms such as actinomyces. DNA microarray analysis helps as the
bacterial genomic DNA often outlasts the viability of the bacteria and a diagnosis can be made
using a small amount of DNA, as opposed to the large numbers of bacteria needed for culture. In
future, an abscess specimen might be sent not for culture and sensitivity testing, but rather for
DNA microarray analysis.
The current scope of microarray applications includes:

. sequencing by hybridization
. resequencing
. mutation detection
. assessment of gene copy number
. comparative genome hybridization
. drug discovery
. expression analysis, and immunoassay (protein microarrays).
Advantages of DNA microarray
DNA microarrays are mainly used for transcriptional profiling but they can also permit large-scale
study of DNA variations, rapid investigation of chromosomal structure and a global identification
of protein-binding sites on DNA.
Disadvantages of microarray
The most significant disadvantages of microarrays include the high cost of a single experiment,
the large number of probe designs based on sequences of low-specificity, as well as the lack of
control over the pool of analyzed transcripts since most of the commonly used microarray
platforms utilize only one set of.
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Microarray by Jamal

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Name:.

Submitted To:. Mam Sidra

Subject:. Adv. In diagnostic technique

Steps involved in cDNA based microarray:

Basic steps of microarray Technology:

The current scope of microarray applications includes:

Advantages of DNA microarray

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