cDNA dilution for efficency calculation - (Nov/21/2008 )
I'm using ddCt method for relative quantification and I want that this value is correct. I know that ddCt calculation is valid if the amplification efficiencies of the target and reference are approximately equal; a sensitive method for assessing if two amplicons have the same efficency is to look at how dCt varies with template dilution. cDNA must be diluted over 100-fold but I don't know if concentration is important. Can I start with 100 ng and then go on with 50 ng, 10 ng, 5ng and 1 ng? Or I have to start with a lower concentration to have good results?
always aim for a dilution range as high as possible eg. 6-7 10fold dilutions. The results are much better. If you have a very abundant target you can use cDNA and a starting amount of 100 ng should be ok.
If you are studying a weakly expressed gene you can switch to artificial standards like serial dilutions of your target sequence inserted in plasmid or simply purified PCR products. You can adjust for the "background" DNA with some inert DNA eg. salmon sperm is often used.
To assess the assay efficiency with a dilution range of only 100fold is not very accurate and chances are high that you are introducing more error than with the ddCt method without validation.