Using Ct values below 10 - (Dec/09/2008 )
I was just wondering if it is acceptable or not to use Ct values below 10 when using the ∆∆Ct method. I have read in various sources that when doing the validation experiment to ignore any Ct values below 10. Does this apply to any run? If so, what is the reason for ignoring Ct's below 10? In one of my qPCR runs, my Ct values are between 8 and 10 and I'm not sure if it's ok to use them. Thanks!
Generally, average of fluorescence intensities of 3-15 cycles will be used, when you set up the baseline.
I really don't see why that you couldn't use Ct below 10 cycles, unless you can obviously see that the baseline is already far above normal.
What is the gene you run to have Ct less than 15?
How much cDNA you used for your real-time?
How low you select your threshold?
As "microlight" has said, fist 15 cycle will be used as baseline; thus if you have Ct less than 15 you for sure use too much template.
The problem for too many templates (Ct less than 15) is repeatability.
If you load same amount template you use now for 4 to 6 wells and select all Ct as you have done, you will find your statistical variation (coefficient of variation (C.V.)) among those wells could be more than 15% and this variation will affect the subsequence quantification (our standard is less than 2.5% C.V.).
My opinion is nothing wrong using low Ct, but if you want a reliable qRT-PCT, you better have higher Ct so the variation among wells will be smaller.