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can contaminating gDNA explain this?


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#1 Kenyon Applebee

Kenyon Applebee

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Posted 24 February 2011 - 09:53 PM

Hello,

I am trying to use qPCR to verify knock-down of genes by RNAi in paramecium. However, I keep seeing enrichment of the gene I am knocking down in the treated vs. the control. The primers are designed to amplify a region of the genes outside of the dsRNA construct, so I do not think that I am seeing signal from that. Additionally, I am generating cDNA using d(T)x primers which should not bind to E.coli-generated mRNA, only paramecium mRNA. I also have some phenotypic observations that suggest the RNAi is working, leading me to suspect there is something wrong with my qPCR.

I have been going over my RNA isolation and RT protocol, and realized that my DNAse is kind of old (about a year). I have ordered some fresh and am prepared to run some no-RT controls on my next plate, but I am scratching my head because while I can see how gDNA could dampen a measured difference in expression, I cannot imagine how it could consistently cause the measured relative expression to change direction.

Is this correct and I need to find another underlying problem, or does someone here see something I am missing?

Thank you for any insight!

#2 Kenyon Applebee

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Posted 24 February 2011 - 10:07 PM

Nevermind folks - after posting this I questioned my assumption that E. coli T7 transcripts are not polyadenylated and found a paper suggesting they are, so Im guessing this is my likely problem. I did not see a way to delete my post, but thanks for reading if you made it this far : )




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