How to remove gDNA from small amounts of RNA - (Nov/06/2006 )

Hi,

I am isolating RNA from cells (approx. 500 000) to do qRT-PCR. But my primers are sensitive for gDNA contamination. I did RNA isolation on columns, but the amount of RNA that I got there was not enough. Therefore I did Trizol and now I have the problem to get rid of the gDNA without contaminating my samples with all kinds of other stuff (like EDTA or DNAse). I did a DNAse I treatment (Invitrogen) but it drastically increased my Cts (by 3!). Therefore I compared it on tissue samples (from which I get large amounts of RNA) with an on column DNAse treatment and found hughe differences. With the on column treatment my Cts were decreased as compared to untreated samples and the no-RTs also did not show any gDNA contamination. I guess it is due to higher starting concentrations of my target RNA as I don't meassure the gDNA with it anymore. The problem is, that I can not use on column treatment with the RNA from my cells as I would lose most of it.
Can anybody give me a good advice on how to get rid of that gDNA without contaminating my RNA?

Thanks a lot!

Just wanted to know, how id you find you that your RNA was contaminated by gDNA?

If you use TRIzol method, from the aqoueus phase that been transfered to fresh tube, you re-extract again by adding TRIzol, BCP/chloroform, then spin again, transfered to aqoueus fresh tube, precipitation by isopropanol, wash with 70% ethanol............

You should be able to get a pure RNA.

Hi

Although I still have not tried it yet, somebody recommended me Turbo DNA free from Ambion, it seems to work well with small amounts of RNA...

Yap,
That is a good product, but the $ also good
I have a wanderful experience using Ambion products