8.1 Microarray experiments  (Page 5/4)

Microarry chips are devices that enable the scientist to simultaneously measure the transcription level of every gene within a cell. Microarrays are commercially available from a number of companies, such as Affymetrix , Invitrogen and Sigma-Genosys , to name a few. The chip is usually constructed by amplifying all the genes within the selected genome, yeast, for example, using polymerase chain reaction (PCR) methodology. The PCR products would then be "spotted" onto the chips by a robot, as single-stranded DNA that is linked by covalent bonds to the glass slide. The spots would be positioned in an array on a grid pattern, where each spot contains many identical copies of an individual gene. A discussion of the chemistry involved in creating a microarry can be found on the technology page of the Affymetrix website. The position of the genes are recorded by spot location, so that the appropriate gene can be identified any time a probe hybridizes with, or binds to, its complementary DNA strand on the chip.

Microarray chips measure transcriptomes, which are the entire collection of RNA transcripts within a cell under the given conditions. To use the chip to measure an experimental transcriptome against a reference transcriptome requires cells grown under two different conditions, the experimental conditions and the reference conditions. The mRNA from the two different conditions are harvested separately, and reverse transcriptase (1) is used to transcribe the mRNA into cDNA. The nucleotides used to synthesize the cDNA will be labeled with either a green or red dye, one color for the reference conditions and the other for the experimental conditions. The microarry chip is then incubated overnight with both populations of cDNAs, and a given cDNA will hybridize with the complementary strand from its gene that is covalently bound to a grid spot on the chip. The chips are washed to remove any unbound cDNAs and then two computerized images are produced by scanning first to detect the grid spots containing cDNAs labeled with green dye, and second to detect the spots contain red-labeled cDNAs. The computer also produces a merged image that will show a yellow spot for grid spots that contain both red- and green-labeled cDNAs, indicating transcripts that are expressed under both sets of conditions. A very nice on-line, animated demonstration of the entire protocol is offered by the Genomics Course on the Davidson College website (2).

In addition to producing a qualitative image that is easy visualize, a microarray experiment yields quantitative data for each spot, consisting of the measured fluorescence intensity of the red signal, the fluorescence intensity of the green signal, and the ratio of red signal to green signal. It is in storing and analyzing the quantitative data that bioinformatics really comes into play in microarray technology. These data sets are incredibly large. For instance, a typical mammalian cell is estimated to have between 10,000 to 20,000 different species of mRNA expressed at a given time.