Expression profiling using cDNA microarrays.
Microarray technology has become increasingly useful in measuring expression levels of a large number of genes and part of a repertoire of functional genomic tools. We describe the methods of cDNA microarray preparation, the use, data collection, and initial data processing. The cDNA fragments are first prepared by polymerase chain reaction (PCR), and then attached to a solid substrate, such as a chemically treated glass slide. Robotic machines spot the prepared cloned cDNA samples in a miniaturized gridded pattern, so that nanoliter amounts of tens of thousands cDNA samples are bound to a single 7.5 x 2.5 cm glass slide. Probes are generated from RNA samples of test and control tissues by incorporating Cyanine dyes (Cy3 or Cy5) in reverse-transcribed products. Probes from a test sample are labeled with one of two Cy dyes and mixed in equal amounts with probes from a control sample labeled with the second dye. The glass slides containing the cDNA microarray are hybridized with the mixed Cy-labeled probes, washed, dried, and scanned using laser scanners with an optimized wavelength to excite each Cy dye. The emission image patterns for each dye are captured by a digital camera using micro-optics and processed into numerical values that positively correlate with quantitative levels of mRNA for each cDNA spot on the slide. The collected data is then further processed, normalized across experiments, and examined via numerous statistical and mathematical approaches to infer changes in expression levels of particular genes due to the treatment tested.