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

, the control RT-PCR primers may be used in a more quantitative manner. Consider following the appearance of the PCR amplification products as a function of cell cycle. Generating PCR amplification products under these conditions will be influenced by primer efficiency and template abundance. Stratagene has determined that each of the primer sets appears to exhibit equal application efficiency, as measured by the rate at which PCR product accumulates during the linear phase of the PCR. This is consistent with the supposition that products generated with these primers reflect the amount of template used in the PCR.

Figure 3

We conducted experiments to evaluate the cycle-dependent accumulation of PCR product for each of the six RT-PCR abundance primer pairs. A single lambda library derived from the liver was used as template in all reactions (Figure 3). The amount of PCR product was measured using quantitative densitometry and signal intensity expressed relative to the cycle number (Figure 4). Based on results using real-time PCR,6,7 we expected the cycle at which product is first detected (the threshold cycle) to indicate the abundance of these transcripts. In addition, we expected the absolute yield of the PCR amplification product to depend on the level of input template. Both of these expectations were consistent with the PCR results we obtained. The more abundant the transcript, the earlier the PCR amplification product was detected, and the greater the amount of PCR amplification product was observed at the plateau. This suggests that these primers may be of utility in semiquantitative measurements of template abundance.

Figure 4

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