I am currently doing some qPCR on some genes (GOI's) that have a very low copy number and to get a consistent signal below 32 Ct's I need to use a dilution of only about 1:3. However at this dilution my HK genes (UBC, HPRT1 and GAPDH) all have very strong expression (<20 Ct's). This means that there is a rather large discrepency between the GOI and HK genes and I now that this can cause variation in the results.
I can obviously solve this discrepancy by using different dilution of the HK and GOI. My question is though will negate the purpose of the performing an internal HK control ... i.e. using different dilutions of the HK and GOI could be analgoues to using different samples?
Thanks in advance for any suggests or help.
Differences between HK Ct values and GOI Ct values
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