Hybridization to Homemade Microarrays

Competitive hybridization of labeled nucleic acids to a microarray is conceptually similar to other hybridization methods, such as Southern blotting. For massively multiplexed microarrays, the adoption of two-color hybridization schemes has been a significant advance. The use of two colors-typically Cy3- and Cy5-labeled nucleic acids-makes it possible to control for factors that affect hybridization intensity, including the number of labeled nucleotides and the T _m of each oligonucleotide. Thus, the difference in intensity among spots on a microarray can be quantified and analyzed to assess biological phenomena, like changes in gene expression or details of transcript structure. This protocol for hybridization is conceptually straightforward-"cold" (nonfluorescent) blocking nucleotide is added to the mixed nucleic acid material, Hybridization buffer is added, and the mixture is applied to the microarray surface. Hybridization occurs overnight, after which the microarray is washed and scanned.