I am doing qPCR based on some samples which have been done by another student. My confusion is that I have some nanodrop for RNA extracted samples and also the nanodrop for cDNA samples synthesized, I know that cDNA measurement are not quite accurate but the thing is that although he has different concentration for RNA samples, his measurements for cDNA are almost similar for all samples, so he told me that he has done his experiments assuming they are all the same and used 4ng/ul {final concentration per 15 ul reaction in his qPCR}.
I would appreciate if you could guide me here.
Some information may be useful:
RT-PCR: 5OO ng RNA in 12.5 ul total reaction
RNA. NanoDrop will detect both newly synthesized cDNA as well as the original RNA, including target (unless your RT method digests it) and all of the other, probably much more abundant, RNA species. Digestion and purification can be done to yield pure cDNA, but for your purposes, this is not necessary. Base qPCR on equal RNA input and include appropriate tests for efficiency and linearity.
RNA. NanoDrop will detect both newly synthesized cDNA as well as the original RNA, including target (unless your RT method digests it) and all of the other, probably much more abundant, RNA species. Digestion and purification can be done to yield pure cDNA, but for your purposes, this is not necessary. Base qPCR on equal RNA input and include appropriate tests for efficiency and linearity.
RNA. If you use nanodrop spectrophotometer to measure NA concentration, you should take into consideration the fact of spectrophotometer's imprecision, especially for cDNA (significant step for cDNA spectroscopy measurements - dNTP removing, which may decrease cDNA yield). For more accurate and reliable results fluorescence methods of NA measurements should be better. You also may combine results of RNA nanodrop spectroscopy and denaturating phoresis, but the best way to conduct research - combination of RNA measurement and normalization to "house-keeping" genes (although may be some problems with "house-keeping" genes (it's another topic).
RNA. The way NanoDrop works it measures all the nucleotide present in a solution. Thus when you measure cDNA on a NanoDrop it will measure the optical density of Newly prepared cDNA, the restover RNA, the nucleotide from the primers. It is generally assumed that the RNA input for cDNA preparation will give almost the same output for cDNA.
I think the important thing is if the amount of RNA for cDNA synthesis was the same. Your comment "5OO ng RNA in 12.5 ul total reaction " seems to suggest that. In any case you will have an internal control, e.g. ref gene, for your qPCRs
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