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RT-qPCR normalization

RT-qPCR normalization

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3 replies to this topic

#1 lqhstephanie

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Posted 30 October 2012 - 10:38 AM

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!

#2 Nabin

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Posted 06 November 2012 - 08:44 PM

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

#3 pcrman

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Posted 08 November 2012 - 10:33 PM

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

#4 Afiqah Alyaa

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Posted 15 November 2012 - 05:07 AM

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 ?




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