Microarrays are the latest tools for DNA profiling used in differential gene expression studies, gene function identification studies, gene co-regulation studies, time- course studies, clinical diagnosis, dose-response studies, identification of biomarkers and many more.
The technique employs three different methods. Spotted cDNA microarrays are used to identify genes of DNA sequences about 500 to 2500 bp long. Spotted oligonucleotides are used extensively in studies involving DNA sequences of 30 to 70 bp. Affymetrix chips are the most precise among them which can cater to the needs of 25 bp long sequences of DNA nucleotides. The choice of each method depends on the nature of the experiment and the length of DNA sequence involved in the study.
The procedure involved in the technology is very simple. The process is based on the principle of hybridization. The concerned gene sequence is isolated and amplified by PCR. The samples are checked for purity using spectrophotometer. Absorptions at different wavelengths 230nm, 260 nm, and 280 nm are used to check for impurities. A260/280 should be ideally greater than or equal to 1.8 for pure DNA samples. A260/280 for RNA samples is approximately 2.0 for those with higher purity. The samples are then printed on with the help of robotic printers to a glass array coated with aminosilanes or poly L-lysine. The coating makes it hydrophobic thus protecting the DNA strands from hydrolysis. It also increases the adherence of DNA on to the slides of microarrays.
The probe is prepared like wise by extraction of nucleic acids from the sample and subjecting them through a similar process involving RT/PCR amplification. The probe is labeled with fluorescent dyes Cy3 or Cy5 and is hybridized with the microarray. After careful washing procedures to remove the unhybridized DNA strands, the slide is scanned with confocal microscopy.
The technique involves excitation of fluorescent dyes with two different wavelengths at the red and green region of the absorption spectra. The result of the two beam excitations are overlapped and produce a combined image.
Suppose you want to find out the differential gene expression patterns of a particular gene under a diseased condition. Then the sample DNA is to be taken from a diseased tissue and the reference from a healthy tissue. Label each of them with a different dye Cy5 (red) or Cy3 (green). On hybridization and scanning, the genes which express under both conditions show equal level of absorption at the two differing wavelengths and hence cancel out to form yellow spot on the developed microarrays. Those with red region then will represent those genes which are over expressed and those with green spots will contain genes which are under expressed. Hence it will be easier for you to identify the target gene by subjecting these spots alone for further analysis.
The Cy5/Cy3 ratio (red/green) is taken as data for further analysis using various statistical tests. The log ratio of the value is a better tool since the distribution of data from the microarrays form a lognormal distribution. A log ratio of 1 for the Cy5/Cy3 value represents the genes which are 1x over expressed (up regulated) and a value of -1 for genes which are 1x under expressed (down regulated). A back ground correction is needed for further accuracy. So the final equation for the data values from microarrays becomes
Log2 Cy5/Cy3 = Log (Red intensity/ Green intensity)
= Log ((Rfg-Rbg) / (Gfg-Gbg)
where Rfg- red intensity foreground; Rbg - Red intensity for background
and Gfg- Green intensity foreground; Gbg- Green intensity for background.
Note that the logarithmic ratio is to the base 2 and not the usual 10. This is because difference in gene expression levels can be as low as small fractions between similar genes or alleles of the same gene. Setting it to the base 2 will help you to identify such minor differences for better identification of the target gene using the microarrays.
There are different scanners employed for scanning the microarrays and producing data for further analysis. Some of the more frequent scanners are LGE Scanner, Codelink Scanner, and Ludwig Scanner etc. The Scanners have inbuilt statistical algorithms to identify the perfect spots for data generation. Spots which are not perfect like an imperfect morphology, contaminated spots etc are marked and not selected for data quantification.
From each spots, approximately 20 readings are taken which makes their entry into the databases of microarrays. The process is a huge one involving three essential steps- Gridding, Segmentation and Intensity Extraction.
Such data bases are in public domain making the task easier for analysis. Microarray Data Analysis is found to be a good tool in almost all fields where the information on these DNA strands is beneficial. One of the most recent applications is the Reverse engineering in which the data is analyzed and used to create the biochemical network pathways of metabolism and gene regulation. Just as the DNA discovery has made a new era in biotech research, microarrays are gaining popularity and extending its applications to newer domains to unfold further mysteries.
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