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Control RT-PCR Primers for Human Gene Transcripts with Varying Abundance


Characterize gene distribution in your cDNA

Brenda Rogers Douglas McKenzie
Stratagene

We introduce six pairs of control RT-PCR primers that are designed to detect specific human gene transcripts with various expression levels. These primer sets provide insight into whether cDNA synthesis has been effective for all abundance classes of transcripts and whether full-length or near full-length cDNA species are present for those genes. The RT-PCR Primers for Genes of Varying Abundance Levels (human) consist of one primer pair each for a high- and medium-abundance gene transcript, and primer pairs for each of four low-abundance gene transcripts. The primer pairs are positioned toward the 5 end of the transcript to provide information about the effectiveness of first-strand synthesis. These RT-PCR primers span intron/exon junction(s) and are designed for use under high-stringency annealing conditions (60C). The primers are designed to have minimal self-complementarity and minimal capacity for self-priming or formation of primer-dimers. To complement the control RT-PCR primers, we also introduce probes for the same transcripts, so analysis of libraries can be conducted using standard probe technologies.

Generating cDNA from RNA is one of the principal methods used in gene discovery and gene expression analysis. While cDNA libraries have always played an important role in conventional screening technologies, they took on added importance in 1991 with the advent of the shotgun single pass, a partial sequencing of entire cDNA libraries.1 This new approach generated large collections of expressed sequence tags (ESTs) that did not correspond to known genes. The impact of this technology on new gene discovery has been enormous.

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