Hi,
I used the SuperScript IV VILO kit for cDNA synthesis and was surprised by the results that NO RT had a higher concentration of DNA than RT, also the concentration of DNA in both RT and NO RT was almost 100 times higher than the concentration of its cognate RNA. As for cDNA synthesis, the RNA is diluted 20 times in the total volume, I wonder how the concentration of synthesized cDNA is higher than its cognate RNA. Can you please advise?
Assuming you're determining [DNA] via spectrophotometry (nanodrop etc), remember that this will also measure all the primers and even free nucleotides you included to make the cDNA synthesis reaction work. Those are all present in excess, and all will contribute to the 260 absorbance.
Do not expect to quantify cDNA this way: assume that RNA is converted at approximately 1:1 (it will, in practice, be lower, but there's nothing you can do about that), and assume that no amplification occurs.
Because no amplification occurs.
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