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CT Cycle Variations in Real-time PCR?
i am not very old with the handling of real time pcr, anyways, i want to ask -
in order to confirm the firmness of my experiment [expression checking] i performed real time pcr using sybr green dye, i want to know is it fine if i follow the following proedure for analysis-
i perform the pcr with the same samples twise using the cDNA from same rna conc[1ug/ul].
then i consider ct values at same threshhold in both the pcr results.
then i select the ct values which are best
what is happening is, there are some ct cycle variations which are greater than 1 or 2 in some cases, in this case i am considering the one which suits best to for my result.... is this fine to do or this is not allowed? please suggest me with your views, i will be very thankful
Last edited by admin; 11-10-2010 at 08:12 AM.
Re: CT Cycle Variations in Real-time PCR?
I think you shouldn't "choose" which one best for you; calculating the average will be better. I prefer to run standard curves (in triplicate), to make sure that the efficiency of your primer match to the efficiency of the internal control you used (GAPDH or beta-actin, etc.). And this can also give you ideas about the variability of your experiment (by doing the triplicate for the standard curve).
But that's just my opinion...
|cycle , pcr , questionplease , realtime , simple , variations|
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