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

w-abundance primer set must be individually determined.

How the RT-PCR Primers May Be Used

Figure 1

When analyzing cDNA quality, we used these control RT-PCR primers many different ways: For example, in Figure 1 and Figure 2, we used a number of the control primer sets to conduct a PCR analysis on lambda libraries derived from several human tissues. We wanted to detect an amplification product, as opposed to evaluate the relative amounts of different transcripts. Consequently, we used a large number of cycles (35 cycles) to maximize the signal. Under these conditions, the plateau effect obscured any quantitative relationship that might exist between the abundance of the transcript and accumulation of PCR product. The results in Figure 1 demonstrate that transcripts for b-actin, g-actin, and protein phosphatase 1 catalytic subunit are present in libraries generated from the lung, muscle, spleen, heart, and liver RNA. Other experiments confirmed their expression in the ovary, testis, cerebellum, and pancreas (data not shown).

Figure 2 shows PCR results obtained with primer pairs for the low-abundance genes ARF1 and ARF3 when tested on lambda libraries derived from the lung and spleen. In every instance, the size of the PCR product was consistent with cDNA, not gDNA, as the source of the PCR signal. These results (Figure 1 and Figure 2 and data not shown) are consistent with the broad tissue distribution of these transcripts and the ability of the control RT-PCR primers to detect their presence.

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