Standard Curve method RT PCR- - (Jul/20/2008 )
Hi,
I have been doing RT-PCR , and in I use standard curve method to quantify. in every PCR plate I run, i was taught that I should run corresponding REFERENCE GENE triplicates. Just not for any given cDNA synthesis, but for every PCR run I included a triplicate of 18S.
However i was recently told that this is a common misconception and that I do not need to do this for every plate, but only for a given cDNA synthesis, and then I can use these reference gene values to normalise genes of interest values.
I would be very gtrateful if any one who is using STANDARD CURVE METHOD COMMENT ON THIS.
thanks.
I have been doing RT-PCR , and in I use standard curve method to quantify. in every PCR plate I run, i was taught that I should run corresponding REFERENCE GENE triplicates. Just not for any given cDNA synthesis, but for every PCR run I included a triplicate of 18S.
However i was recently told that this is a common misconception and that I do not need to do this for every plate, but only for a given cDNA synthesis, and then I can use these reference gene values to normalise genes of interest values.
I would be very gtrateful if any one who is using STANDARD CURVE METHOD COMMENT ON THIS.
thanks.
I try to only run each gene once, including housekeeping genes. However, if I do need to perform multiple runs, I make sure that I use a calibrator sample (or two), usually my standards, to make sure each run is consistant. I think running your housekeeping gene in every run is a massive waste of money.
hi
well if you run your standards for every run i don't see what is the problem for an extra triplicate for your housekeeping. the point of standard curve method is comparing every Ct to the standard run in each run. if you want to save money, you should try Pfaffl method, especially if you do gene expression.
best.
Chris