How to eliminate DNA contaminaiton during RNA extraction? - (Jul/17/2006 )
I am using SDS-PHENOLE method isolating RNA, But I always get DNA during my RNA product. In my hands it seems that using acid phenole/chloroforme(5/1, twice) can not eliminate DNA. Someone suggested a modified method to reduce DNA contamination, the improved method is discribed in Nucleic Acid Research, 1993m vol. 21 N 8 2019-2020. The modification involves the addition of a brief RNA selective precipitation step following the lysis of the cells with the guanidinium thiocianate solution. This is achieved by the addition of 1/3 volumen of 95% ethanol and incubation of the mixture on ice for 5 minutes. Following centrifugation for 15 minutes the supernatant is removed and the RNA pellet dissolved in 1/2 of the original volume of guanidinium thiocianate solution.
I am not very clear about the operation procedure, and I also do not know the mechanism it works. I think the remants of the lysed tissue will coprecipitate with the RNA , I doubt if the deposites will contaminate the RNA and result in a reduced RNA yield. If someone has tried the protocol, please notify me. Any answer or idea will be appreciated. Thanks!!
Why not use DNAse? We use the RNeasy kit from Qiagen. And one step of the procedure is the use of DNAse, which you can also order seperately: RNAse-Free DNase Set (cat.no. 79254).
I second the use of DNase to clean up RNA preps. Its very dependable too so there's little doubt as to whether you have residual DNA or not.
edit: make sure its RNase-free DNase. I've tried to use old Dnase in the past that may have become contaminated with RNase and ended up with samples containing neither RNA nor DNA...
Nucleic acids will generally coprecipitate if you are not careful or your buffers are off. The accepted method of getting rid of DNA is using DNase as the other members described.
If your lysis buffer contain guanidium thiocynate do not use buffered phenol use acid phenol in the extraction
acid phenol pH 4.5 saturated with water or sodium citrate buffer pH 4.5.
the DNA still in the acid orguanic phase (phenol-chloroforme) and you will get RNA in aquous phase after phenol chloroforme extraction.
i would also suggest DNase. It's only active with a metal ion, so you can control with chelating agents.