How can recombinant forms of a gene be used to measure gene expression?

One of the most popular applications is to compare expression of a set of genes from a cell maintained in a particular condition (condition A) to the same set of genes from a reference cell maintained under normal conditions (condition B). First, RNA is extracted from the cells. Next, RNA molecules in the extract are reverse transcribed into cDNA by using an enzyme reverse transcriptase and nucleotides labelled with different fluorescent dyes. For example, cDNA from cells grown in condition A may be labelled with a red dye and from cells grown in condition B with a green dye. Once the samples have been differentially labelled, they are allowed to hybridize onto the same glass slide. At this point, any cDNA sequence in the sample will hybridize to specific spots on the glass slide containing its complementary sequence. The amount of cDNA bound to a spot will be directly proportional to the initial number of RNA molecules present for that gene in both samples.

Following the hybridization step, the spots in the hybridized microarray are excited by a laser and scanned at suitable wavelengths to detect the red and green dyes. The amount of fluorescence emitted upon excitation corresponds to the amount of bound nucleic acid. For instance, if cDNA from condition A for a particular gene was in greater abundance than that from condition B, one would find the spot to be red. If it was the other way, the spot would be green. If the gene was expressed to the same extent in both conditions, one would find the spot to be yellow, and if the gene was not expressed in both conditions, the spot would be black. Thus, what is seen at the end of the experimental stage is an image of the microarray, in which each spot that corresponds to a gene has an associated fluorescence value representing the relative expression level of that gene.