[MatthewHaas]

I have used Turbo DNase followed by cleanup on Qiagen columns. The Turbo DNase kit does provide an inactivation agent that is supposed to remove the enzyme, but time and again I find that the inactivation agent fails to adequately remove the enzyme (I have been using it for ~ 5 years now). For more routine uses, I'll use the inactivation agent, but for my more valuable samples, I always follow up with cleanup on a column.

This thread is rather old, so I'm not sure if I'll be able to get a response, but here goes...

When you do the column purification to remove the DNase enzyme, do you add buffers (which buffer/what volume?) to your RNA sample before passing it though the column and proceed with the same protocol you used initially to extract the RNA? (With Qiagen Mini kit: RLT, RW1, RPE.) I need to remove some DNA from my RNA samples and I'm a bit concerned that you said the inactivation agent isn't completely removed. I want to make sure the cDNA created from my RNA libraries isn't automatically digested. Thanks for your input.

[DStephens@NuGEN]

Hi MatthewHaas,

The Qiagen RNAeasy kits include separate protocols for RNA Clean-up after DNase treatment. All you need to do is bring your sample volume up to 100 uL with nuclease-free water, add RLT and EtOH as directed, and add to the column. See p. 54 of the RNeasy Mini Handbook here: https://www.qiagen.com/us/resources/...a33e24&lang=en

[Amar]

That's really interesting thanks DStephens@NuGEN. I never knew the RNAeasy columns can be used for clean up.

MatthewHaas: The turbo DNase kit is fantastic and in my experience the inactivation buffer works great. Just be sure to stay well away from the inactivation pellet. I have also been told that DNAse is extremely fragile and sensitive to heat. cDNA synthesis includes a heat-inactivation step, which should hopefully degrade DNAse. But as DStephens@NuGEN said, if the samples are precious then perform a cleanup step.

Good luck!

[kumard]

Has anyone used RNAzol, from Molecular Research Center, they make the claim that DNAse is not required after extraction. See the link: http://www.mrcgene.com/rnazol.htm

Dave

Its a very old thread, but let me add that I am using RNAZol RT and I see DNA contamination even after following their protocol. I am struggling with this problem. I talked to their tech support and they informed me to do DNAse I treatment. The advantages that RNAZol RT have is that you can use water for the phase separation and the smallRNA fraction profile is better (on small RNA sizing on Agilent bioanalyzer) than Trizol (with RNA of RIN 9). I hope it will help someone.