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Enough checks for gDNA contamination in qPCR?


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3 replies to this topic

#1 sevendoctor

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Posted 27 June 2012 - 01:25 PM

Hi Everyone,

I have been doing qPCR measuring transcrpt expression in a eukaryote cDNA and am worrying about possible gDNA detection in my qPCR.

I dont DNAse my RNA prior to reverse transcribing to cDNA, but my primers are on different exons. In addition, I only get a single peak in my melt curve analysis, and when I sequenced my product it was the cDNA sequence, not the gDNA sequence. Is this sufficient to exclude possibility that gDNA contamination may be interfering with my qPCR? Is there other checks that I should be taking?

Thanks for any help you can give!

#2 leelee

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Posted 27 June 2012 - 06:31 PM

Do you run a no RT control?

#3 Curlis

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Posted 28 June 2012 - 07:29 AM

Hey,

Did you run gel after isolating RNA? If you run the gel you will be able to any DNA contamination in your RNA.

I think this is the best way to see any genomic contamination in RNA.

#4 sevendoctor

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Posted 28 June 2012 - 11:27 AM

Thanks for the replies.
I did run all the RNA on a gel but couldnt see a gDNA band.
I havent run a No-RT control. I could do this, but was hoping to avoid this as I've got so many samples to qPCR. Perhaps if I run one or two No-RT with all my primers?




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