MRNA and 18S–RNA coapplication–reverse transcription for quantitative gene expression analysis

Abstract

Fluorescence-based reverse transcription real-time quantitative polymerase chain reaction (RT–QPCR) is a highly sensitive method for the detection and quantitation of mRNA. To control and correct for sample variability, some common housekeeping genes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin, and ubiquitin are often used as endogenous standards. Other internal calibrators such as 18S–ribosomal RNA (18S–RNA) have also been used, but further methodological concerns arise given that ribosomal RNA lacks the 3′ poly-A tail typically associated with messenger RNA. To take advantage of the constant expression levels of 18S–RNA and the precision of oligo-(dT) primed first-strand synthesis, we have developed a method that combines oligo-(dT) with an 18S–RNA-specific primer in the initial reverse transcription (RT) reaction. This strategy, termed coapplication reverse transcription (Co–RT), allows for the analysis of multiple target genes with the advantages of 18S–RNA normalization from a single RT reaction. In this article, we describe Co–RT and present tissue distribution and expression level analysis of several target genes using this method. Co–RT provides increased sensitivity and higher accuracy than do the standard random primed RT methods.