PCR data analysis if the efficiencies aren't equal
Posted 24 June 2009 - 03:20 AM
Now I am trying to perform the real-time quantitative RT-PCR (TaqMan) for the detection changes in gene expression some gene involved in drug resistance of ovarian cancer patients. For comparison the changes of expression in cancer tissue I use pool of the samples from normal ovarian tissue (my calibrator sample). As endogenous control I select 18S RNA. The real-time PCR reactions are performed by Applied Biosystems 7300 Real-Time PCR System. And I have some problem with my experiment. First, CT 18S RNA is very high (close to 9-11). Second, I have positive meaning CT from no template control (NTC) sample and control sample without RT. In this case Ct meanings are low (33-38), but I think CT meanings should be absent. Contamination problem is excluded by repeating analysis with a new set of reagents.
What is the matter? Should I change my endogenous control and dilute sample?
Also I tried to use comparative CT method (ΔΔCt), but validation experiments didnít show that the efficiencies of the target and endogenous control amplifications are equal. May be, someone can give advice about the methods of data analysis gene expression where standard curve and equal CT for target and endogenous control are unnecessary.
Thanks in advance!
Posted 24 June 2009 - 05:28 AM
Also, if the efficiencies of the target and endogenous control primer sets are different, you should use the Pfaffl correction to take the efficiencies into account rather than ΔΔCt, which is only an approximation technique and is only applicable when efficiencies are very similar (many users ignore this).
See the paper: Pfaffl, M. (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research 29 (9): 00-06
Edited by Dr Teeth, 24 June 2009 - 05:31 AM.
Science is simply common sense at its best that is rigidly accurate in observation and merciless to fallacy in logic.
Thomas Henry Huxley
Posted 24 June 2009 - 08:31 AM
Of course the Pfaffl method corrects for this.