RT-qPCR normalization - (Oct/30/2012 )
I'm new in RT-qPCR.
So I isolated my RNA and did a Nanodrop Spec on the RNA ng/uL. Then I did reverse transcription using Taqman box. Our lab usually do 500 ng RNA per reaction (18uL). But one of my RNA samples only had ~32 ng/uL, so I didn't dilute it. Now my final RNA/rxn for this sample is ~303 ng, while other samples are all ~500ng.
Then I did qPCR on the cDNA I got. I plotted the relative abundance.
Is there anyway to normalize the qPCR data because I started with different amount of RNA during RT? Or is the relative abundance already normalize the difference for me?
And what is relative expression level?
I think once you normalize with your reference gene expression, your amount of RNA should be normalized automatically.
It is very important to start with the same amount of RNA. Too much difference in starting RNA cannot be normalized by internal controls.
Do you suggest after we dilute the RNA samples to a standardize concentration, we should actually check back the concentration with Nanodrop, before subjecting the samples for cdna synthesis ?