How to convert the Ct values to copy number - (Sep/08/2008 )
How can we convert the Ct values abtained in q RT-PCR to copy numbers.Say if i have got Ct value for 1st sample at 28 and for the 2nd sample at 30.How much copy number difference is between sample1 and sample2.
You will have to generate a standard curve with different dilutions of the cDNA sample. Keep all other reactions constituents exactly the same as your reaction condition. Plot the cDNA concentrations against the c(t) time. Run the reactions in triplicates or more to obtain better CV values. You can only compare across samples with the same cDNA and primer set.
Ahh that's simplicity itself!
Work out the efficiency of the reaction (another topic entirely), then use the Pfaffl method.
where e is the efficiency of the reaction, control is the Cp value for your control, and sample is the Cp of your sample. Of course you should control with a housekeeping gene (or 3+) so your equation would be divided by
e^(control - sample) for the housekeeping gene (or the geometric mean of these if you are using multiples).
for example, assuming perfect reaction efficiency (2), your control Cp=28 and your unknown Cp=30
2^(28-30) == 0.25. That is, your unknown has 1/4 (or 1/2^2) the concentration of your control.