Protocol Online logo
Top : New Forum Archives (2009-): : PCR, RT-PCR and Real-Time PCR

Amount of cDNA input in RT-PCR - (Jan/17/2017 )



My understanding and what I have always done for my RT-PCR is to use the same amount of cDNA for my GOI and housekeeping genes and then compare the expression. I've read somewhere that you can use different dilutions of cDNA for the GOI and HK if the HK's Ct value comes up very early. Is this correct? Can you do this? And also how do you correct for this when you're doing your calculations?


I had always use the same quantity of cDNA for both. If the HK ct plots are too early I change it to other, always use a HK that is somewhat similar to your GOI instead of forcing the HK to match your GOI.