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Normalization of qRT-PCR following nuclear/cytoplasmic fractionation


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#1 NemaToStella

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Posted 03 September 2013 - 03:09 PM

Hey guys, it's been a while...

and I'm guessing this is a very trivial question although I can't seem to wrap my head around it:

in order to determine the subcellular localization of my favourite RNA (i.e. nuclear vs. cytoplasmic; I am reluctant to do FISH) I am going to fractionate my cells and isolate both nuclear and cytoplasmic RNA.

Now here's where I am unsure: I'd like to simply do a relative quantification, for instance to determine that RNA X is three times as abundant in the cytoplasm vs. the nucleus. Which gene would I use for normalization, if that is even possible?

 

Thanks a lot!

 



#2 PhalanxBio

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Posted 03 September 2013 - 03:28 PM

I suggest picking a group of candidate control genes and explicitly testing whether they are equally expressed in the nucleus vs cytoplasm.  When you find at least one gene that is equally expressed in the two fractions, then you can proceed to normalize your genes of interest.



#3 NemaToStella

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Posted 03 September 2013 - 03:37 PM

Thanks for your reply, PhalanxBio! So I would reverse transcribe equal amounts of nuclear and cytoplasmic RNA and then run a qPCR with my control genes, hoping the Ct values of nuclear and cytoplasmic samples are roughly the same? Is that the idea?



#4 Tabaluga

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Posted 09 September 2013 - 02:19 AM

So I would reverse transcribe equal amounts of nuclear and cytoplasmic RNA and then run a qPCR with my control genes, hoping the Ct values of nuclear and cytoplasmic samples are roughly the same? Is that the idea?

 

That should be fine. Try several possible housekeeping genes and use the best ones, at least three (the more the better), for normalisation.


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#5 NemaToStella

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Posted 23 September 2013 - 01:29 PM

Thanks Tabaluga! I appologize for the late reply...






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