Direct Cyanine-dUTP Labeling of RNA for Microarrays

Abstract

This is the simplest method to label RNA for use in expression analysis. RNA is reverse-transcribed using both oligo(dT) and random hexamers as primers. The random hexamers improve overall efficiency of labeling, especially at the 5′ end of the RNA. Fluorescently labeled dUTP is incorporated into the cDNA. After reverse transcription, the RNA is degraded, and the labeled cDNA is purified from unincorporated Cy dyes. Finally, samples labeled with Cy3 and Cy5 dyes are mixed and combined with blocking nucleotides and used for hybridization.