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RT-PCR procedures

RT-PCR can be performed as either a two-step or an one-step procedure. Each has certain advantages: 1. Two-step procedures A. Two tube, two-step procedure. In the first tube, first-strand cDNA synthesis is performed under optimal conditions, using either random hexamers, oligo(dT) primers (generating a cDNA pool), or sequence-specific primers. An aliquot of the RT reaction is then transferred to another tube (containing thermostable DNA polymerase, DNA polymerase buffer, and PCR primers) for PCR. Advantages of this approach: This method is useful for experiments where multiple transcripts have to be analyzed from the same RT reaction or for specific applications such as Differential Display Reverse Transcription (DDRT) or Rapid Amplification of cDNA Ends (RACE). Also, since the RT reaction is performed under optimal conditions, this approach produces the longest RT-PCR products (up to 14 kb in length, if the appropriate enzymes are used). B. One tube, two-step procedure. In the first step, reverse transcriptase produces first-strand cDNA in the presence of Mg2+ ions, high concentrations of dNTPs, and either specific or non-specific [oligo(dT)] primers (reaction volume, 20 l). Following the RT reaction, an optimized PCR buffer (without Mg2+ ions), a thermostable DNA polymerase, and specific primers are added to the tube and PCR is performed. This approach may be useful when template amounts are limited, since the entire RT reaction is used in the subsequent PCR.
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