Transfected Cell Microarrays

Essay by zyncodexUniversity, Master'sA+, February 2004

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Microarrays are slides "printed" with thousands of small spots of biological material, in a mechanism similar to inkjet printing, which allow rapid assaying of many cell phenotypes under different conditions. Traditional DNA-based microarrays are printed with DNA oligonucleotides complimentary to the template strand of the full compliment or a subset of genes from a given organism, which are then UV-crosslinked to the slides. mRNA is isolated from the organism in question under two different conditions (i.e., different developmental stages, growth conditions, genotypes, etc.). The two samples of mRNA are then reverse transcribed in the presence of fluorescently-labeled dNTP substrates (different label for each sample; i.e, green and red), forming a number of fluorescently labeled cDNAs whose concentration is proportional to the original corresponding mRNA concentration in the organism. The fluorescently labeled cDNAs are then used to probe the microarrays and the unhybridized probe is removed. DNA oligonucleotide spots that fluoresce green under UV light indicate that the corresponding mRNA was present in higher concentration in the "green sample" relative to the "red sample."

This is because the cDNA probe with the higher concentration should bind the complimentary printed oligonucleotide to the exclusion of the other probe. The observance of a green spot therefore indicates that it is likely that in organisms under "green sample" conditions, the gene corresponding to both the oligonucleotide and cDNA is upregulated relative to the organisms under "red sample" conditions. The reverse holds true for observance of a red spot, and observance of a spot intermediate in color between green and red indicates that the gene is expressed at the same concentration in both samples. Therefore, using oligonucleotides corresponding to the full compliment of genes from a given organism, the RNA phenotype and thus the level of expression of each gene can be measured under many...