DNase digestion and RNA degradation - Cancer tissue RNA isolation (Jan/06/2005 )

Hi,

I want to describe a probleme we always run into with RNA from animal and human tissue.
We use and tested different methods of RNA isolation from tissues. The Qiagen columns work well and the on column DNase digest helps to eliminate DNA cotamination, but....
We are not always able to use the columns and have to isolate the RNA different with Trizol or GTC.
The RNA has a good quality and the amounts are reasonable. But DNA contamination is detected by RT-PCR.
We tried several DNase digestions after RNA isolation and that results in a big loss of the amount and degradation of RNA.
That ocours in about 80% of all samples.
Does anybody recocnize the same problems and has anyone a solution?

If you read what GeneHunter Inc. claims, everyone's DNase is contaminated with RNase except theirs. This is, in their assessment, a major cause of RNA degradation. You could try some of theirs, but I think they're full of crap.

When isolating from tissue you are more likely to have residual RNase contamination from the tissue. You could do multiple extractions to remove as much residual protein (including possible RNAses) as possible.

What method do you use to precipitate the RNA following the treatment? If your method is inefficient, that could explain some of the loss. I suggest using NH4OAc/isopropanol precipitation. I have had very good recoveries with this method. There are protocols for it on this site.

Why are you unable to use RNeasy columns with animal tissues; I have done this successfully on many occasions.

Good luck! May the Lab God's smile upon you!


- The Biology Ninja