RT-PCR Control Primers?
carnwath at tzv.fal.de
Thu Jun 2 10:53:28 EST 1994
I would like ask about experience with control primers for RT-PCR, other
than those for beta-actin.
When RT-PCR primers do not flank an intron, it is not possible to
distinguish between a PCR product produced from reverse transcribed mRNA
(cDNA) and a product produced from genomic DNA contamination. In other
cases, where there is no PCR product, it is important to verify that the
reverse transcription step worked before concluding that there was no
Primers for beta-actin which flank an intron in that gene have often been
used as control primers. They produce a large PCR product from genomic
DNA and a small PCR product from cDNA. However, there are some problems
with beta-actin primers. Several contributors to this group have pointed
out that the presence of actin pseudogenes gives positive signal for the
small product when reverse transcription really didn't work. On the other
hand, the sensitivity for contaminating DNA is low (the two copies of
genomic DNA per cell must compete with 1000 cDNA copies for amplification)
which makes it necessary to have a separate control reaction where the
reverse transcriptase is either missing or heat inactivated).
Would anyone care to share the sequences for a primer pair that can be
used as an RT-PCR control in a wide variety of cell types? I would think
that some housekeeping gene which was transcribed at a low but constant
level would be best. We are working with murine and bovine RNA samples.
Thanks for your comments,
Joe Carnwath carnwath at tzv.fal.de
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