Arbitrarily Primed PCR of RNA

If a single primer is to be used as both upstream and downstream primer for amplification, cDNA can be synthesized with this single primer or with an oligodT primer to select for polyadenylated transcripts. Using the same primer

From: Methods in Molecular Biology, Vol. 97: Molecular Embryology: Methods and Protocols Edited by: P. T. Sharpe and I. Mason © Humana Press Inc., Totowa, NJ

Fig. 1. The two major approaches that can be taken for the random amplification of RNA. In the first case (A), synthesis of the first strand can be driven with either oligodT or the same arbitrary primer that will be used for amplification. After random amplification, the product obtained should be identical in both cases. In the second case (B), the downstream (3') and upstream (5') primers are different. The first strand of cDNA is synthesized with a tailed oligodT that will only recognize a fraction of the mRNA population. Random amplification is then performed with a second arbitrary primer. Note that three different products can be obtained depending on the primer combination (see ref. 16).

Fig. 1. The two major approaches that can be taken for the random amplification of RNA. In the first case (A), synthesis of the first strand can be driven with either oligodT or the same arbitrary primer that will be used for amplification. After random amplification, the product obtained should be identical in both cases. In the second case (B), the downstream (3') and upstream (5') primers are different. The first strand of cDNA is synthesized with a tailed oligodT that will only recognize a fraction of the mRNA population. Random amplification is then performed with a second arbitrary primer. Note that three different products can be obtained depending on the primer combination (see ref. 16).

for both the cDNA synthesis and the PCR is very convenient, since only one primer is required and good matches with the primer will be already selected during cDNA synthesis. On the other hand, priming with oligodT will select for polyadenylated transcripts, and thus, cDNA will not be made efficiently from ribosomal or heterogeneous nuclear RNAs, which are extremely abundant as compared to mRNA. A disadvantage of the latter is that nonpolyadenylated mRNAs will not be represented among the cDNA population. After oligodT-driven synthesis of cDNA, a single primer might then be used for the amplifications. This method also allows the use of nested primers that will select for population of PCR products that are in low abundance and thus underrepresented when a single primer is used (5,6). The basis for this approach is discussed below.

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