I perform alot of RT-PCR using Relative Quantitation. I look at the relative expression of a gene between samples using an endogenous control to normalise the expression between the samples.
For cDNA synthesis it is recommended to normalise the RNA quantities used among the samples, i.e. having the same RNA concentration in each RT reaction. However, I do not perform this as I use endogenous control expression to assess cytotoxicity between samples and RNA normalisation would lead to equal endogenous control expression, thus distorting cytotoxicity results.
Of what I know is that RNA normalisation is important to eliminate differences in RT-PCR efficiencies. When I compare Ct expression between biological controls normalised with my endogenous control, I see little difference (~0.2 Ct). Even among absolute expression of biological controls there is not a huge difference in Ct (this for cells that have been growing for 3 days in separate wells). Among biological replicates there is max 10% difference in RNA concentration when assessed for yield.
What is your opinion on this? Should I continue as I do, normalise RNA concentration among biological controls or among all samples?
RNA quantity normalisation. Important for Relative Quantitation RT-PCR?
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