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Canīt get rid of DNA contamination in RNA

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#1 Katuss



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Posted 26 February 2015 - 12:58 AM

Hi everyone, 


I need a help with my RNA samples. I isolated RNA from mice organs using Trizol. The concentrations of RNA are high, about 1000-5000 ng/ul in 80-100 ul. RNA is quite clear, the ratio is 1,9-2,1. So I performed the treatment using 1ug of RNA with 1U of DNAse (Novagen) in final volume 10 ul. I usedbuffer for M-MLV Reverse Transcriptase (Promega). I did PCR for GAPDH (from 1ul of treated RNA) and unfortunately, the contamination of DNA was high. So I tried a new protocol and used 5-10 U of DNase for 1ug of RNA in final volume 50 ul, then I cleared the RNA using phenol-chloroform and it didn´t help. The DNA was still there and there was no difference between 5U and 10U of DNase. 


For some samples I used RNeasy Quiagen kit to isolate RNA with the DNA removal colums. However, when I put 1 ug of RNA and 1U of DNase to treat these samples, the DNA was mostly still there, I performed PCR for GAPDH and I had strong bands. So I tried to use 10U of DNase for 1ug RNA in 50ul final volume and then clear RNA using EZ-10 Spin Column RNA Cleanup and Concentration Kit (Bio Basic). Then I performed PCR for GAPDH and the DNA is still there. 


I don´t know what to do, I am thinking about buying a new DNase (Ambion) a try this one. Did I do something wrong ? Do you have similar experiences ? Thank you very much for your advice.

#2 Katuss



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Posted 26 February 2015 - 03:41 PM

I want to ask one more question. Can I treat the samples (isolated RNA containing contaminating DNA) with DNase and subsequently repeat the reisolation of RNA with Trizol ? 


And next question is, can I try to digest DNA with endonuclease such as RsaI ? Is it safe for RNA ? Are the endonucleases (or buffers for endonucleases) inhibitors in qPCR ? Is it necessary to wash these samples prior to qPCR ?


Thank you

#3 merlav



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Posted 02 March 2015 - 12:11 PM

DNase is a very delicate enzyme that a can denature easily like for example mixing with vortex.  My advice buy a new DNase and mix  gently never vortex. I use the RNeasy kit and the one that have the DNA removal columns the capacity of removal is  20ng of DNA more or less, so I digest with dnase (I had used: sigma ampd1, ambion  dnase turbo and rnase free dnase kit from qiagen all 3 are really good products and very easy to use).  Do a double DNase digestion.  Clean all with a rnase/dna removal like rnase away or rnase zap.  

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