Dear all,
I am currently working on a reverse-transcription protocol (otal RNA extracted from CHO cells, then reverse-transcribed to be used in qPCR) with QuantiNova Reverse Transcription kit supplied by Qiagen.
When performing a negative control (ie., no template RNA), I obtain approx. 2 µg/µL DNA. I measured out the RT mix alone (containing oligo-dT, random primers and dNTPs), diluted 1:5 (4 µL in a total reaction volume of 20 µL when performing RT reaction), and obtained 3 µg/µL DNA.
I would like to know how to determine the cDNA concentration of my samples, as far as components of the RT mix are also absorbing (without performing a blank with RT mix each time).
Many thank for your help,
Lucie
Hi,
I am wondering why you might need to know the concentration?
It's fairly well accepted that trying to measure cDNA concentration after the RT is massively inaccurate as you've also found. It is generally assumed that you get roughly 1:1 conversion of RNA to cDNA (although this is also highly variable).
As this is the case, it's more usual to normalise the amount of RNA being used in the RT reaction rather than attempting to measure afterwards.
However, this is not actually required either. If you are using multiple endogenous controls (housekeepers) in your qPCR experiments, these should allow for normalisation of input RNA/cDNA (in the Delta Ct step of analysis) and so there should be no real advantage in knowing your input cDNA concentration.
I usually advise a minimum of 3 housekeepers in any experiment since more HKs = less variability and you can drop any that are not stable across your sample set.
Hope that helps.
Ben.
You do NOT need or you dont have to do this step. However, you can measure the cDNAs for all samples before running the rt pcr. Also u can test the Ef as well.
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