I am trying to convert RNA to cDNA in order to quantify gene expression. I started with 50 ng/uL RNA and I obtained 1500 ng/uL nucleic acid at the end (I am not if it's only cDNA or cDNA+remaining RNA). That does not seem normal to me since it is supposed to be a conversion not an amplification.
What you measured was a mixture of RNA-cDNA and nucleotides..Let's suppose you used 1000ng of total RNA for cDNA synthesis, and your RT-final volume is 50 ul then the concentration of your cDNA is about 20ng/ul.
Karima Ben Mansour basically nails it, here. You don't spec cDNA because this gives meaningless data.
cDNA synthesis involves
1) RNA
2) dNTPs
3) primers (oligodT, random hexamers/nonamers)
4) cDNA (eventually)
And even though it's 4) that you want, 1-3) are still there, and will still absorb strongly at 260, giving wildly inaccurate quantification.
Assume the conversion is 1:1.
Based on my experience when converting RNA to cDNA, there is no change in the concentration of cDNA. What is read after the reverse transcription process is the total concentration of the solution consisting of cDNA, RNA residues or even reagent residues. So the concentration remains the same between RNA and cDNA.
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