anfortin at webnet.qc.ca
Mon Jul 24 18:30:59 EST 2000
I have to do semi-quantitative PCR in order to study the regulation of
expression of receptors mRNA. I've already done optimization of annealing
temp., MgCl2 conc., etc...
If I want to compare the level of two different mRNA in response of a
stimulation of the cells, which of the following methods is the right one ?
1. Search for the optimal cycle number of primers pairs for each receptor
studied, stimulate the cells, isolate RNA and do the RT-PCR with optimal
cycle number (which can be different for both primer pairs) for each
receptor and stimulation in parallel with the GAPDH std for each different
Finally, doing the ratios signal/GAPDH and comparing the ratios to see up or
down-regulation of the receptors.
2. For each conditions of stimulation, do PCR at 25, 30, 35, 40 cycles (for
example). If a PCR product appear at a lower cycle number compared with an
other, it means that the corresponding mRNA is more abondant. With this
method, if its OK, how can I use the GAPDH std ? Ratio for each cycle
number? I have already start to determine the optimal cycle number for GAPDH
and although it is not evident (because there is variability, difficult to
see the plateau of the curve in order to use the cycle number in the linear
portion), I obtained signals of different intensities versus cycle number.
So, in my mind I can't use a "variable" standard !!!!
This method seems to be time consuming, on the other hand, if after the
stimulation of the cells I get for example a very strong response (PCR
signal), I will be in the plateau of the PCR amplification curve even if I
have predetermine the optimal cycle number!!!!
Any suggestion ???
Thanks a lot
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