Differential Gene Expression Analysis by Combining Sequence-Tagged Reverse-Transcription Polymerase Chain Reaction with Pyrosequencing
Abstract It is an important way to understand the gene function by relatively comparing gene expression levels among different tissues or cells. For the moment, most of the methods for gene expression detection are based on dye labels. To establish a novel approach without using a dye label, a sequence-tagged reverse-transcription PCR coupled with pyrosequencing (SRPP) was proposed. In this technique, the gene from a source is labeled with a source-specific sequence by sequence-tagged reverse transcription (RT). Then PCR on the pools of each source-specific RT product was performed, and the source-specific amplicons were decoded by pyrosequencing. In the pyrogram, the sequence represents the gene source, and the peak intensity represents the relative expression level of the gene in the corresponding source. The accuracy of SRPP was confirmed by real-time quantitative PCR. Finally, the relative expression levels of the Egr1 gene among the diabetes model mice, obesity model mice, and normal mice were successfully detected. In comparison with real-time quantitative PCR, the advantages of SRPP include dye-free detection, inexpensive instruments, and simultaneous comparison of a given gene expressed in multiple sources.