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DNase removal prior to mRNA isolation - (Nov/21/2008 )

Colleagues,

Please I would like you suggest me some methods to remove DNAse I from my total RNA prior to mRNA isolation. Do you think it is necessary to purify again with one chloroform wash or just isopropanol precipitation would be enough???

I isolated total RNA using a TRIZOL-based protocol.

Thanks in advance for your suggestions,

Danilo

-Danilo Eduardo-

QUOTE (Danilo Eduardo @ Nov 22 2008, 05:34 AM)
Colleagues,

Please I would like you suggest me some methods to remove DNAse I from my total RNA prior to mRNA isolation. Do you think it is necessary to purify again with one chloroform wash or just isopropanol precipitation would be enough???

I isolated total RNA using a TRIZOL-based protocol.

Thanks in advance for your suggestions,

Danilo


If you extracted using Trizol, technically you don't even need DNase treatment if you separate the aqueous layer correctly. how do you isolate mRNA?

-chrisbelle-

QUOTE (chrisbelle @ Nov 23 2008, 01:51 AM)
QUOTE (Danilo Eduardo @ Nov 22 2008, 05:34 AM)
Colleagues,

Please I would like you suggest me some methods to remove DNAse I from my total RNA prior to mRNA isolation. Do you think it is necessary to purify again with one chloroform wash or just isopropanol precipitation would be enough???

I isolated total RNA using a TRIZOL-based protocol.

Thanks in advance for your suggestions,

Danilo


If you extracted using Trizol, technically you don't even need DNase treatment if you separate the aqueous layer correctly. how do you isolate mRNA?



Hello chrisbelle,

I am going to isolate mRNA using the MicroPoly(A)Puristâ„¢ Kit (Ambion, Cat. # AM1919). This kit is based on a binding solution along with Oligo(dT) Cellulose. This mixture is incubated with continual rocking or shaking, allowing hybridization between the poly(A) sequences found on most mRNAs and the Oligo(dT) Cellulose. The Oligo(dT) Cellulose is then transferred to a Spin Column and washed to remove nonspecifically bound material and ribosomal RNA. Finally, the poly(A) RNA is eluted using pre-warmed THE RNA Storage Solution.

I do think DNAse I treatment of total RNA is necessary prior to mRNA isolation as I did a preliminary trial using untreated-DNAse total RNA to isolate mRNA. When using this mRNA as template to amplify intronic sequences to check gDNA contamination, I got an amplicon, which suggests mRNA contains DNA debris. I carried out this experiment using a PROMEGA kit based on oligoDT binding and magnetic beads.

Thanks again for your suggestions,

Danilo

-Danilo Eduardo-

Hmm, the Ambion kit should be specific enough to extract only mRNA, not genomic. Anyways if you wanna do DNase treatment after total RNA prep I would say do the phenol chloroform again but your yield may be much less. Actually do you mean you want to remove the DNaseI or DNA from your total RNA? I don't think its necessary to remove DNase I itself.

-chrisbelle-

Can your DNase be heat inactivated?(For ex the one which we use from NEB can be inactivated by heating to 75C for 10min). Even after heat inactivation still do you want to clean up your sample? Will using RNA isolation column be helpful in that. We clean up our RNA sample isolated using Tri by Qiagen RNeasy columns whenever it is necessary.

-Calvin*-