I am trying to quantify the level of viral RNA in mouse brains following infection using reverse transcription quantitative PCR. I have made a standard curve for determining the quantity (in ng) of viral RNA from Ct value by serially diluting in vitro transcribed viral infectious clone RNA. Using my equation, I can determine ng of genomic RNA and then convert to copy number using the FW of the target. Also, I run a known quantity of genomic RNA each time so I know that the standard curve works.
My question is, how does normalization factor into this? I don't think I can use the standard curve to determine copy number from normalized Ct. Do I need to make a standard curve of my endogenous control? Do I need to use an endogenous control at all or can I just report at copy number/ng RNA?
I don't think the endogenous control really factors into it for quantitative RT-QPCR, does it? If you want to know the mean viral genome copy number per cell, then you need an endogenous control, but that would have to be a DNA control and you'd have to do a standard curve for that too I believe.
See the bottom of the attached file for some possible ideas.
Always a good question.
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