Random failures with RT-PCR

KUN Hematol. Lab. kunazhe3 at caos.kun.nl
Thu Feb 23 07:52:43 EST 1995

Since more than a year we are performing RT-PCR for the detection of mRNA
of growth factors and extracellular matrix proteins without a lot of
problems. However, since several weeks we have problems with the
reproducibility of PCR within and between experiments. Performing PCR with
the same cDNA and mastermix, same primers, same experiments, gives in a
number of amplifications a negative result. Using different reactions
vessels or different thermal cyclers did not give a solution. Also working
with purified mRNA instead of total RNA did not work out. In addition the
DNAse teatment was checked but also this did not solve our problem. Still
it appears that randomly within one experiment certain PCR reactions fail
while the same reactions with the same compounds in a neighbouring
reaction vessel is succesfull. Could anyone give us a clue about the
mechanism of this problem and how to overcome it.

				Peter M. van der Kraan
				Dept of Rheumatology
				University Hospital Nijmegen
				The Netherlands

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