Is it necessary to treat total RNA with DNAse I when isolating mRNA? - (Jan/30/2006 )

I'm with John

I find that my yield and purity are both better if I do not treat with DNase...but you have to design your primers properly and I use melting-curves with my qPCR. I suppose it depends on your application and your RNA prep kit, but I find that there is very little/no gDNA contamination. According to Ambion, it's essentially impossible to find a DNase that has no contaminating RNase and this will adversely affect your work.

but, this is only my opinion and many people swear by DNase treatment

Another thing about DNase digestion is that the enzyme does not remove the DNA but breaks it down into smaller fragments. Even if you can't see it on a gel, you may still be able to amplify fragments <100bp very efficiently

But as Aimikins says - horses for courses

So, what RNA prep do you guys use that produces less than ten to twenty copies of genomic DNA contamination? Trizol? Filter Columns? Magic Magnetic Beads?

I'd love to see these beautiful gels and qPCR results (w/no RT treatment control) that you are producing with no DNase treatment.

-Matt

If you are smart and make your amplicon 200-300 bases (as it should be), this is obviously not an issue.

DNase works...the reason why it reduces your "yields" is because it's cutting up the genomic DNA in your prep, hence decreasing your "yield" of genomic DNA in your RNA/DNA mixture.

-Matt

I agree that DNase works and I often use it

The RNA prep method I use is still in development and is proprietary to my company so I can't tell you what it is, but it doesn't use DNAses and you will see it released in the summer

Just for you, here's a gel pic of 8x 1ug RNA, with 20ng of gDNA in the final well



These samples show mean % gDNA contamination as 0.03% by qPCR against a standard curve of gDNA - that's 300pg DNA per ug RNA

Hi colleages,

Thank you very much for your suggestions, I will take them into account.

However, I will perform my mRNA isolation with no DNAse treatment of my total RNA as I suppose that my kit will only capture by means of magnetic separation from total RNA, the mRNA.

Once I have my results, I will post them.

See you then,

Danilo[i]

Sorry, I forgot to tell them that I will use my mRNA to do RT-PCR (reverse transcriptase-PCR) to turn it in cDNA, cloning it and then use it for cDNA microarrays.

On the other hand, I agree that DNAse is a cheap treatment to remove DNA contamination from RNA. Nevertheless, the bottle-neck remains in the way how to stop the DNAse activity since most methods to do that (EDTA chelation, heating, proteinase K digestion/phenol:cloroform extraction) cause most times the RNA become degraded.

I am enclosing my total RNA gel run on a 1.5% denaturing agarose gel . As you can see, there is no visible DNA contamination. However, as you know, virtually all RNA isolation procedures always results in DNA contamination not easily visible.

The fisrt five lanes contains 25, 4, 5 and 3 ug of RNA; the next ones contain twice.

I used a phenol:cloroform/LiCl-based protocol, not a kit to isolate total RNA.

Danilo