title: Preparation of fluorescent-dye-labeled cDNA from RNA for microarray hybridization.
abstract: This protocol describes how to prepare fluorescently labeled cDNA for hybridization to microarrays. It consists of two steps: first, a mixture of anchored oligo(dT) and random hexamers is used to prime amine-modified cDNA synthesis by reverse transcriptase using a modified deoxynucleotide with a reactive amine group (aminoallyl-dUTP) and an RNA sample as a template. Second, the cDNA is purified and exchanged into bicarbonate buffer so that the amine groups in the cDNA react with the dye N-hydroxysuccinimide (NHS) esters, covalently joining the dye to the cDNA. The dye-coupled cDNA is purified again, and the amount of dye incorporated per microgram of cDNA is determined.
authors.forename: Manuel
authors.initials: M
authors.lastname: Ares
mesh_terms: D018076:DNA, Complementary / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D005456:Fluorescent Dyes / Q000378:metabolism*; D046228:Microarray Analysis / Q000379:methods*; D009693:Nucleic Acid Hybridization / Q000379:methods; D012313:RNA / Q000235:genetics; D048348:Reverse Transcription; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D018076:DNA, Complementary; D005456:Fluorescent Dyes; D012313:RNA