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
volume, 20 l). Following the RT reaction, an optimized PCR buffer (without
ions), a thermostable DNA polymerase, and specific primers
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
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