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

ion and cDNA length are particularly important for low-abundance mRNAs, as most new genes are likely to be discovered from this population of transcripts.

DNA hybridization studies indicate that the typical mammalian cell expresses between 10,000 to 30,000 mRNA species at any one time. These mRNAs can be grouped into three distinct abundance classes.4,5 Most prevalent are the approximately 10 mRNAs that account for about 10% of the overall transcripts. The rare transcripts, approximately 10,000 distinct mRNA species present at between 1 to 15 copies per cell, account for about 45% of the total mRNA in the cell.

Features of the Gene Targets

We used several criteria to select target genes for the Stratagene set of control RT-PCR primers: a fairly ubiquitous and constitutively expressed gene transcript, different sources of RNA expressed in a fairly uniform manner, and some known information about the abundance levels for the transcript. We applied an arbitrary classification of transcript abundance that was consistent with the original studies of Bishop, et. al.4 In general, we designated high-abundance genes as those expressed at approximately 1% of the total RNA, medium-abundance genes as those expressed at 0.1 to 1.0%, and low-abundance genes as those expressed at equal to or less than 0.1%. This corresponds to approximately 3000 copies/cell, 300 to 3000 copies/cell, and less than 300 copies/cell, respectively. Using these criteria, it was fairly straightforward to identify suitable high- and middle-abundance gene targets. In contrast, most low-abundance gene transcripts were not ideal candidates because of their somewhat limited tissue expression patterns. To solve this potential problem, we developed primer pairs for several different low-abundance gene transcripts, each of which exhibited broad but not universal tissue expression. Hence, choosing the appropriate lo
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