Detection of Genes on the Chip

To estimate the relative expression levels of each gene within a particular cell type or tissue, total cellular RNA is extracted by standard techniques and reverse transcribed into radiolabeled (for example, with 32P or 33P-UTP) or flu-orescently labeled (for example, with Cy-3 or Cy-5-UTP) cDNA probes. These probes are then hybridized to the chips. Fluorescently labeled probes are more commonly used as they are convenient and quantitative and can be measured with more precision using commercially available equipment.

After the hybridization of the probes, slides are scanned by laser. The laser causes the excitation of fluorescently labeled cDNA probes. Only the spots representing mRNAs in the sample give emission signals. The emission is measured using a scanning confocal laser microscope, and the fluorescence data at each and every spot are analyzed by appropriate software. The absence of fluorescence in a specific spot means that complementary mRNA is not present in the sample. If the fluorescence is present, the intensity of the signal obtained is a measure of the level of particular mRNA in the examined cell population.

Usually an investigator wants to compare mRNA abundance between two different samples (or a sample and a control). In this case, cDNAs from the sample and a control are labeled with two different fluorescent dyes (e.g., red label for the cDNAs from the sample and a green label for the control), and these two cDNA populations are allowed to hybridize to the same microarray slide. If a particular mRNA from the sample is in abundance, the spot with a complementary probe will be red; if the concentration of the particular mRNA is higher in the control, the spot will be green. If both samples contain the same amount of a given mRNA, the spot will be yellow. Thus one can draw conclusions about the relative expression levels of the genes based on the colors and fluorescence intensities of the microarray spots. However, some commercially available systems permit the hybridization of only one sample per chip. In this case, data from one chip are compared with data from other different chips using appropriate software. Thus, the relative expression levels of genes in one cell type and another cell type can be compared directly.

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