Muhammad Umair Mustafa

Roll No. 1041 (Morning)

Microarray is one of the most recent advances being used for cancer research; it provides
assistance in pharmacological approach to treat various diseases including oral lesions.
Microarray helps in analyzing large amount of samples which have either been recorded
previously or new samples; it even helps to test the incidence of a particular marker in tumors.
Till recently, microarray's usage in dentistry has been very limited, but in future, as the
technology becomes affordable, there may be increase in its usage. Here, we discuss the various
techniques and applications of microarray or DNA chip.

Prevention, diagnosis, and treatment in dental practice are based on an understanding of the
biology of underlying oral health and disease. Few aspects of patient care will remain untouched
by today's rapid advances in biological research. In the future, dentists may use inexpensive but
remarkably sophisticated diagnostic tests to diagnose infection, oral lesions, and symptoms of
temporomandibular dysfunction (TMD). The small variations in the DNA sequence that lead to
different characteristics (such as skin color, facial features, or height) are known as
polymorphisms, which also can cause or contribute to the development of many syndromes and
diseases. These genetic variations can be easily identified by the microarray technique.

Microarray provides a basis to genotype thousands of different loci at a time, which is useful for
association and linkage studies to isolate chromosomal regions related to a particular disease.
This array also can be used to locate chromosomal aberrations related to cancer, such as
segments of allelic imbalance, which can be identified by loss of heterozygosity. By comparative
genomic hybridization techniques on genomic DNA, amplified or deleted regions in the
chromosomes can be identified, such as in the case of oral cancer.

Technique

Gene microarray technology rests on the ability to deposit many (tens of thousands) different
DNA sequences on a small surface, usually a glass slide (often referred to as a “chip”). The
different DNA fragments are arranged in rows and columns such that the identity of each
fragment is known through its location on the array. Two types of microarrays are gene
expression microarray and tissue microarray (TMA). Techniques like Northern blot and reverse
transcriptase-polymerase chain reaction (RT-PCR) allow testing for only a few genes per
experiment. But microarray or “global expression profiling” not only looks at orders of
magnitude more genes than was possible previously, but also has the advantage that the genes
examined are not influenced by preselection of genes.

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. The remaining DNA fragments are washed away. The target DNA pieces can be
identified by their fluorescence emission by passing a laser beam. 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.

The study of the expression of most, if not all, genes in a specimen is not hypothesis-driven as
most of the studies used to be, but is instead referred to as “discovery-type research” or in a less
flattering description as “fishing expeditions.” Whereas cDNA derived from a tumor is
hybridized to a chip to study gene expression levels, alterations in DNA copy number (gene
amplification or deletion) can be measured by hybridizing fluorescently labeled DNA from a
tumor specimen to these chips. TMAs are constructed by transferring cores of paraffin-
embedded tissue to pre-cored holes in a recipient paraffin block. Over 500 cores can be placed in
a single block by this technique. Sections cut from TMA blocks can then be used for
immunohistochemistry (IHC) or in situ hybridization studies. TMAs are similar to gene
expression microarrays in having samples arrayed in rows and columns on a glass slide; they
differ in that each element on the TMA slide corresponds to a single patient sample, allowing
multiple patient samples to be assessed for a single molecular marker in one experiment, while
gene expression arrays allow assessment of thousands of molecular markers on a single patient
sample per experiment.

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.

Gene microarrays have been used for comparative genomic hybridization. In this technique,
genomic DNA is fluorescently labeled and used to determine the presence of gene loss or
amplification. Array-based comparative genomic hybridization (aCGH) has been used to map
genetic abnormalities in a wide range of tumors, including breast carcinoma, bladder carcinoma,
fallopian tube carcinoma, gastric carcinoma, melanoma, and lymphoma. Gene expression data
can identify groups of cases with significantly different outcomes where routine histopathologic
examination does not permit subclassification.

The conversion of a non-invasive tumor to an invasive tumor also warrants research. Clark et al.
studied the gene profile of melanoma cells which became metastatic, and found a gene, RhoC, to
be expressed more in metastatic cells than in non-metastatic melanoma cells. Microarray-based
expression profiling allows us to identity families of genes as well as the important molecular
and cellular events that might be important in complex processes like metastasis. Practical
applications in future include diagnostic and prognostic management of patients. Clinicians will
be able to use microarrays during early clinical trials to confirm the mechanisms of action of
drugs and to assess drug sensitivity and toxicity. They can be used to develop a new molecular
taxonomy of cancer, including clustering of cancers according to prognostic groups on the basis
of gene expression profiles. Areas that can be coupled with microarray technologies include
classification of diseases, or molecular phenotyping; the study of gene function in relation to
gene regulatory networks, or functional genomics; pharmacogenomics and developmental
biology.

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.

Early detection of oral precancerous lesions

Leukoplakia or white lesions of the oral cavity may result from a myriad of reversible conditions.
Currently, microscopic examination fails to identify the small subset of these lesions that
progress to oral cancer. Identification of gene expression profiles or “genomic fingerprints” will
allow clinicians to differentiate harmless white lesions from precancerous lesions or from very
early cancer. Recent studies have illustrated the effectiveness of microarrays in oral cancer. In
future, samples taken from an incisional biopsy or brush biopsy may be sent to a laboratory for
gene expression analysis. Early diagnosis and management of oral cancer is correlated with
increased survival. Identification and treatment of premalignant and early cancerous oral lesions
may become one of the most valuable services in future performs.

Conclusion

This review has given a small outline of the technique behind microarray and the various steps
involved. The technique, though limited at present in its applications due to the cost factor, may
widen its prospects once there is increase in the availability of commercial products. The ability
to record large number of old samples and analyzing them for various genetic alterations helps in
understanding the concept of molecular biology. Microarrays hold much promise for the analysis
of diseases in the oral cavity. Classifications of oral disease by DNA, RNA, or protein profiles
will greatly enhance our ability to diagnose, prevent, monitor and treat our patients. Currently,
microarrays are primarily a research tool. Microarrays promise a more biologically based,
individualized, and vastly improved standard for oral care, which will have great clinical impact
on the way dentistry will be practiced in the future.