Hello Diego Llamazares,

maybe the simplest way would be trying another DNase (e. g. you can ask colleagues from lab next-door). This let you know, if the problem is in DNase or something other. Also, small part of residual DNA could be bound to residual proteins and such proteins could shield it against cleavage, theoretically.

But I am interested in this sentence you wrote: " I have obtained samples with around 10ng / ul of RNA and 0.5ng / ul of DNA. " How did you find out? When I do similar treatments with DNase I simply believe, that DNA was successfully cleaved :-) Please let me know about your method.

Good luck

Martin

Hello Martin,

Thanks for your answer. I presume that the DNase should be OK because I just purchased it. I am going to do a test with clean DNA samples to check if it works.

When I talk about concentrations I refer to RNA and DNA concentrations obtained just after extraction doing the kit, measured with QUBIT. Then, after performing the DNase treatment, I still get the same DNA concentration.

What do you think I could do to get rid of such proteins? Using a protease?

Bests

Diego

Hello Diego,

thanks for answer, QUBIT sounds great, in our lab we don´t have such instrument, we routinely use Nanophotometer for spectroscopic measurement of isolated nucleic acids, but unfortunately it cannot distinguish between RNA and DNA.

I think you may try proteinase K before DNAse treatment (theoretically, I have never done this before). But be sure, that you have inactivated added proteinase before you subsequently add DNAse :-)

Also DNAses are inhibited by EDTA, if your buffers contain EDTA, the DNAse will be inhibited.

Good luck

Martin

Hi Diego,

I had similar problems with DNase treatment of RNA extracted from corals. Did you use the troubleshooting steps mentioned in the manual of DNase kit? They recommend a second round of DNase treatment. "According to a recent publication†, RNA samples from tissue or cells purified with column-based methods may contain significant DNA contamination—as much as 20–50% of the prep. The study found that the nucleic acid content of samples varied widely from consisting of nearly pure RNA to containing mostly DNA. Samples with DNA contamination that remains after completing the DNA- freeTM Kit procedure may benefit from a second round of rDNase I treatment". It goes like this, "For this second treatment, add 0.15 volumes 10✕ DNase I Buffer and 1–2 μL rDNase I to the sample, and incubate at 37°C for 20–30 min. After the DNase digestion, follow the standard inactivation procedure starting at step 3. For RT-PCR with double DNA-free- treated RNA samples, we recommend limiting the volume of treated RNA to 20% of the final RT-PCR volume". I guess it will help. Probably you can use RNA Clean up kit (MN or Zymo) after first DNase treatment so that the second treatment works well.

Also, DNase is a very sensitive enzyme, and sometimes even rigorous mixing or pipetting can inactivate it. So I agree Martin. Try using another vial or get it from another manufacturer.

In my experience, LiCl further complicates stuff. Its difficult to get rid of residual DNA after LiCl treatment.

Hope it helps. Best wishes!

Thanks Martin and Gaurav for your answers!

I think I managed to solve the problem. What I did was just isolating RNA using the traditional LiCl method for tricky tissues like mine (pine cambium) available here (https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602007000400003&lng=en&nrm=iso&tlng=en).

Whereas the miRvana kit gave extremely low and bad ratios in Nanodrop, the traditional method gave ratios of around 1.9 (260/280) and 2.3 (260/230) which seem pretty good to me. I measured one sample in QUBIT (45ng/ul RNA and 5 ng/ul DNA) and after half an hour of DNase treatment the DNA concentration was 3ng/ul.

This means that in my case the problem is the kit that cannot purifiy clean RNA.

I did try a second round of DNase but did not work either. I will try it again now with the clean samples as going from 5 to 3 ng/ul is not a great degradation.

Thanks again!

Hi Diego,

if the RNA downstream application is RT-PCR, you can use a RT kit from Clontech : PrimeScript™ RT reagent Kit with gDNA Eraser, it works very well for me