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qPCR, RT- Control


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#1 crystalviolet

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Posted 16 April 2010 - 06:37 AM

Hi,

I am trying to do a qPCR to test if my stable overexpression is successful,
However, the RNA isolation kit that we're using does not necessarily eliminate all the genomic DNA (even though it includes a DNAse digestion)

Normally, I make sure to include some introns when I design primers to eliminate any problems, but the problem with overexpression clones is that they include a copy of open reading frame in their genomes, so there are no introns.. (and the genomic DNA contamination problem is very apperant)

So, I thought I could do a qPCR to compare parental cells and overexpression clones together with their RT- controls. In the RT- controls, I obtained some Ct values, But I'm not sure how to normalize those. (Normally I normalize them to Gapdh)

Do you think it is a good idea to normalize RT- controls to the Gapdh Ct values of RT+ samples, & calculate fold changes with respect to the parental cell lines?

I'm completely lost :S

#2 tea-test

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Posted 16 April 2010 - 07:01 AM

hi,

you can try a DNase digestion of the RNA that is prepared just for reverse transcription. this is how i get rid of the transfected plasmid DNA in transient transfected cells when i want to look at the expression on the RNA level. maybe its also better to use less RNA as input material like 250ng instead of 1g, so you have also less DNA that has to be digested. you don't need much cDNA just to check the expression anyway....
of course, don't forget the RT- controls.

i used this one, very simple protocol:
http://products.invi...Search-Product#
tea-test: The artist formerly known as Ned Land

#3 crystalviolet

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Posted 17 April 2010 - 09:36 AM

Thanks a lot for the tips :lol:




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