We had the following
A human blood sample had RNA concentration of 142,85 ng/ul, we used 3,43 ul in 20 ul (with a mass of 490 ng) using the iscript kit. Nanodrop reading for this blood sample after reverse transcription :1500ng/ul
A human brain sample had RNA concentration of 75,7 ng/ul, we used 6,4 ul in 20 ul (with a mass of 490 ng) using the iscript kit. Nanodrop for the human brain sample after reverse transcription :540 ng/ul
Hi
Just by nanodrop or spectrophotometer reading it may not be feasible to estimate quantity of the cDNA. It would be more appropriate to dilute the samples (should have similar integrity/quality) first to the same concentration and then go for RT. This will, in a way, ensure some normalization (wrt to RNA quantity) during cDNA synthesis. Minot variations later can be addressed using ref genes (if the analysis involves qRT-PCR)
bye
Best is to 1st quantify your RNA and use same amount of RNA for cDNA synthesis..sorry but never heard if some one looks for amount of CDNA after synthesis and as Kubista mentioned.. it can give your lots of false positive values.
Dear Jean Neil
You can of course test the efficiency of your cDNA synthesis using an artificial spike as Mikael Kubista suggests, but this only give a hint of your amplifiable cDNA amount as it does not take into consideration how degraded your samples might be. It is also correct that using the Nanodrop or similar UV based absorption methods are not very precise. I would however suggest that you quantify by qPCR. If you have cDNA of one of your genes of interest of known quantity, you can include this (or these) as a reference. I would make a dilution series of your cDNA to be able to establish efficiency of your qPCRs and if possible include references of similar length and known concentration in the same run. At the end of the day, the concentration we often want to know is that of the amplifiable cDNA and that is best obtained by doing amplification. Consider to quantify the longest and shortest ampliqons of your interest, to account for sample degradation.
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