qPCR

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rachawilson
rachawilson's picture
qPCR

Hello all-

I have not been doing qPCR very long but hopefully you all can help me.  I am working with a NFkappaB luciferase reporter and transfecting/co-transfecting into my cells.  Unfortunately my mentor does not like the whole measure the lucfierase activity....i still to do this day do not understand why because this is the way most if not all scientists do.  Anyways so I have been trying to look at emergence of fluoesense (luciferase) by qPCR with my normalizing factor being 18s rRNA.  Honestly to me this seems a little silly because not all my cells are taking up this plasmid and normaling to this endogenous gene does not make sense to me.  The NF kappa B plasmid does have hygromycin resistance gene on it.  I was thinking about making primers for this and using this as my internal standard.  Does this sound good to anything?  Is this a silly idea?  Please help---any advice would be grateful.

Thanks-

rachawilson

Ivan Delgado
Ivan Delgado's picture
 

 
To be honest I do not blame your mentor because luciferase activity assays can be very deceiving. Of course, like any application, it depends a lot on how well the assay is designed and used, so it can be as good as qPCR. 

To your question, I do think that using your hygromycin gene as the internal standard is the better choice for the reason you describe. Having said this, anytime I am trying to normalize qPCR assays, it is a good idea to test different internal standards. Furthermore, I would even run the 18S assay too, just to see what results you get when you normalize against 18S versus hygromycin.

Good luck.