DNase I treatment of RNA sample - (Aug/25/2005 )

Hello,
I need to use DNase I to treat a RNA sample contaminated with genomic DNA. I have DNase I from ambion and its 10X buffer. That kit doesn't have a stopping solution which inactivates DNase I and eliminates divalent cations. So I wonder how can i use that kit? I know that DNase I can be inactivated heating 5' at 75, or perform a phenol-cloroform re-extraction. I need to know your experience (your protocol with a similar kit). Thanks a lot

Hi,

you have to add EDTA to stop the reaction prior heating followed by ethanol precipitation. The EDTA will chelate the divalent cations.

Freiberger

Which concentrarion of EDTA do you use ?
Can you perform reverse transcription from the solution with EDTA ?

i usually treat my samples (cell lysates) with DNase I before RNA extraction (TriReagent)

Seb_

Hi,

is the Trireagent the same as Trizol? So at what point do you treat your sample with DNaseI?

First you lyse the cells, then you treat them with DNaseI and then you add the Trireagent (or Trizol?) -???

@Marvilla: ~1.5mM EDTA, I think RT-PCR should work. I'll tell you in about a week... after I've done my first RT-PCR ;-).

Freiberger

Hi,

I am in the same situation as Marvilla.
I do not know, even it might be too obvious, does ethanol precipitation remove EDTA from the solution, so that I will not have to care if it affects or not a posterior PCR reaction?
So what concentration is the minimum for EDTA?
I am using Trizol reagent and i am also interested in the protocol for DNase I treatment before RNA extraction. Could anyone give as some light???

Thanks

coco

TRI reagent is ambion's version of trizol. they are virtually the same. Both are based in the use of guanidinium isothiociante as denaturanting agent. It seems to me that DNA contamination is inherent to this method and DNase treatment is anavoidable for aplications such real time pcr.
I am in doubt how can you treat your cells with DNase I before RNA extraction with Tri reagent.
I asked directly to ambion how to use DNase I. They told me the same as you, inactivate it with EDTA (5mM instead 1.5mM) and heating 5min at 75. The problem is how to get rid of EDTA to perform subsequent reverse trancription. I believe RNA precipitation can slow EDTA content but not elimitate it. I finaly decided to perform a second RNA extraction with trizol. Tomorrow I will know if it works.

ok Marvilla, so you mean that after the DNase reaction you add EDTA, heat 5 min at 75 and then add again 1ml of Tri reagent and do the whole protocol for RNA extraction again? is this what you mean with performing a second RNA extraction with trizol??
Please let us know how did it work.

coco

-cell lysis:
Tris, EDTA, SDS and NaCl

-DNase I treatment of latter crude extract:
10 U/mL (Sigma) 30 min at 37°C
and heat-inactivation 10 min at 70°C

-subsequent RNA extraction:
using classical TriReagent protocol

Voilà

Seb_

Coco: It is exactly what I am going to do.