3 replies to this topic

[lqhstephanie]

Hello all,

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?

Thank you!

[Nabin]

I think once you normalize with your reference gene expression, your amount of RNA should be normalized automatically.

[pcrman]

It is very important to start with the same amount of RNA. Too much difference in starting RNA cannot be normalized by internal controls.

[Afiqah Alyaa]

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 ?