mRNA and 18S-RNA coapplication-reverse transcription for quantitative gene expression analysis

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.