Is it necessary to treat total RNA with DNAse I when isolating mRNA? - (Jan/30/2006 )
Please I would like to know whether it is necessary to treat my total RNA with DNAse I prior to isolation of mRNA using a PROMEGA kit (Cat. No. Z5300). Since the mRNA isolation kit is intended to take off Poly( A)+mRNA tail from total RNA, I think the DNAse tratment can be avoided. What do you think???
Thank you in advance for your advices.
I think that you should use a lot of DNase.
You'll have more DNA than RNA from that prep, probably...depending on what tissue/cell type you are isolating.
You shouldn't need to DNase if you've used that particular kit because it uses magnetic separation to isolate your mRNA from other nucleic acids (via streptavidin-coupled magentic beads and biotinylated oligo[dT] primers).
You can go ahead and DNase it, but you'll just be increasing your chances of introducing RNases and/or other contaminants.
If you want to perform any kind of meaningful expression analysis (i.e. RT-PCR, microarrays), you will want to use DNase. You probably wouldn't be able to publish your results otherwise because they will be inconsistent for those applications.
We are talking about single-copy detection systems here...it doesn't take much.
Good point. I suppose it really does depend on what his/her downstream intentions are.
If you want to continue with your RNA and that contains an intron, you can easily distinguish between reverse transcribed RNA and amplified DNA due to the intron. Then you wouldn't need DNase, otherwise you better use it.
So, I agree that it depends on the application.
Even if it does contain an intron, it will make your SYBR qPCR look ugly...there are lots of reasons why you want to use DNase. Isn't DNase pretty cheap, guys (I use Ambion)?
Agreed Mistic, I was just talking about amplification of mRNA to clone or to detect its presence, not about qRT-PCR.
In my experience, when isolating mRNA from a pool of total RNA, DNase treatment is unnecessary due to the poly A selection method employed
I think most people here are missing that part of the question
I personally don't believe any method to be 100% selective, so I believe that you will always have some DNA contamination. Even after DNase treatment you will probably have some left, but it will be less...