In qRT-PCR experiment, we first optimize the concentration of first strand cDNA to 25ng/μl in each sample. We use 1 μl cDNA template in 20 μl reaction mixture subjected to qRT-PCR experiment, for which template concentration must be strictly equal in each sample under study; then we have to determine the number of PCR amplification cycles required for the fluorescent signal to cross the threshold (i.e. exceeds background level) as the actual "raw data" in qPCR are the ct values (cycle threshold). Hence we equalize the concentration of first strand cDNA to 25ng/μl in each sample.

Thank you. So how do I ensure 25ng/uL in each sample. With 1uL cDNA my samples does not have equal cDNA concentrations which makes it difficult to determine the difference in gene expression among them.

Hello Toluwase,

I would suggest that you add systematically RNA electrophoresis on agarose gel after extraction to ensure that you have good RNA integrity, as nanodrop is not an optimal tool to check the integrity of RNA.

In addition, maybe you can use a more sensitive method to detect the exact concentration of RNA.

Finally, using stable housekeeping genes have the role to normalize your target RNA transcript expression, and as long as the difference between the expression of the samples' housekeeping gene is less than 1 Ct you can use them.

Keep in mind that there may be always a little difference in the concentration between the samples, which is due to some slight error in manipulation (pipetting..).

Sincerely,

Y.S.