Detection of antisense RNA transcripts by strand-specific RT-PCR.

Abstract

Comprehensive genome annotation requires extensive cDNA analysis. This analysis has identified natural antisense transcripts (NATs), which are distinct from the microRNAs, siRNAs, and piRNAs, in a number of diverse eukaryotes. This wide conservation supports the possibility of an important role for NATs in regulating cellular processes. Investigating their roles requires the confirmation of expressed sequence tag (EST) data and the detection of antisense transcripts in distinct cellular backgrounds. This chapter describes the use of a reverse transcription polymerase chain reaction (RT-PCR) method for the detection of antisense transcripts. The protocol was designed to reduce the number of first strand synthesis reactions during screening for antisense transcripts through the utilization of antisense directed primers and oligo dT to prime first strand synthesis. These results are further confirmed using sense and antisense directed primers in first strand synthesis. Results indicate that optimization of the screens requires proper controls to confirm removal of gDNA contamination and to rule out self-priming as a source of first strand products.