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DNA contamination from adenovirus afted DNase treatment

RNA adenovirus extraction dnase dna

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#1 juelraja

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Posted 26 September 2013 - 01:12 AM

Hi,

I extract RNA with trizol from cells infected with eGFP adenovirus. RNA extraction is done with trizol, so we always use DNase to get rid of possible DNA contamination. With adenovirus we keep having positive eGFP signal with RT-qPCR in NORT controls. So all DNA should be cleaved with DNase 1, extracted RNA is not translated to cDNA but we still end up having eGFP with RT-qPCR (SYBRgreen chemistry). We use actin as a reference gene and we do not find actin in these samples, only eGFP.

 

So it seems that DNase 1 is not able to cleave adenoviral DNA or DNA is inside capside when DNase treated. This is done after Trizol treatment so I would think that the capside brokes down during this but...?

Does anyone have any experience on this?

Thanks!



#2 PhalanxBio

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Posted 26 September 2013 - 08:57 AM

I'm guessing your pipets, work area, buffers, etc. are contaminated.



#3 juelraja

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Posted 30 September 2013 - 04:16 AM

That was the first thing we also thought but we can repeat these results and other negative controls (etc no template control) are always blank so we dont belive it is contamination...







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