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.