Dye-free gene expression detection by sequence-tagged reverse-transcription polymerase chain reaction coupled with pyrosequencing.

Presently most techniques for gene expression analysis are based on a dye label. Here we describe a novel method for comparing gene expression levels among various tissues or cells by sequence-tagged reverse-transcription PCR coupled with pyrosequencing (termed "SRPP"). This method includes three steps: (i) reverse transcription of mRNA with sources-specific RT primers consisting of a tail at the 5'-end for supplying a common PCR priming site, a source-specific sequence in the middle, and a poly-T stretch plus several degenerate bases at the 3'-end for annealing the mRNA strand. (ii) PCR amplification of the templates produced by pooling sequence-labeled cDNAs equally from different sources. (iii) Decoding and quantification of the source-specific sequences tagged in the amplicons by pyrosequencing. The signal ratio in the pyrogram is proportional to the amounts of mRNAs among different sources. As the signal is detected by observing bioluminescence, neither dye, nor electrophoresis, or laser source was used. The expression levels of six kinds of genes (Cdk2ap2, Vps4b, Fas, Fos, Cdk4, and Actb) among the kidney, the brain, and the heart tissues of a mouse were accurately detected, suggesting that the new method is promising in quantitatively comparing gene expression levels among different sources at a low cost.