Microarrays for Gene Expression Profiling: Fabrication of Oligonucleotide Microarrays, Isolation of RNA, Fluorescent Labeling of cRNA, Hybridization, and Scanning

Publisher Summary A DNA microarray is an orderly arrangement of usually thousands of DNA spots typically less than 150 gm in diameter that provides a medium for hybridization of labeled nucleic acids extracted from biological samples. Oligonucleotide microarrays are usually printed on slides with an activated surface that allows end-point attachment of 5’- or 3’-chemically modified DNA. The common theme is that the glass surface of the microarray slide is coated with active chemical groups, which facilitate the binding of end-modified DNA, typically amino modified. Oligonucleotide arrays have several advantages over cDNA microarrays. Gene-specific oligonucleotides can easily be designed and synthesized so there is no need for verification of bacterial clones and high-throughput production of polymerase chain reaction (PCR) products. The quality of RNA for microarray experiments must be of high quality. It should be intact and contain no genomic DNA or enzyme inhibiting substances. Several extraction methods have been tested with a wide variety of biological samples. TRIzol has become our method of choice because it gives consistent, reliable results and is considerably less expensive than kit-based products.