Validation calculations - (Dec/12/2008 )
When calculating the amplification efficiency of target and reference primers, the literature says both should be more or less equal if wanting to do relative quantification: ie a plot of dCt vs long conc should have slop of less than 0.1
My question is: is this figure absolute? Ie, are negative values OK to use too? My amplification efficiencies using two different reference primers are -0.18 and -0.15... are they still suitable for relative quantification?
The deltdelta-Ct method simplifies by assuming the same amplification efficiency for all primers.
"... it has been reported that PCR efficiencies of target and reference amplicons can vary over a range of 1.8–2.0. When this variation in PCR efficiency is taken into account, it can easily be calculated that a real 10-fold difference can turn up as any value between 0.7 and 210. Even with a 0.04 range in PCR efficiencies (from 1.78 to 1.82) already a 4-fold error in fold-difference will occur...." (Ramakers et al., Neuroscience Letters 2003).
But you can use the Pfaffl method (Pfaffl et al., Nucleic Acid Research 2001) that allows a realtive quantification corrected for a primer specific efficiency.
Hope this helped.