This is the best solution and its so fast and easy!! https://www.thermofisher.com/order/catalog/product/AM1907

The columns can be the same, but the buffers are different.

Hi Larissa

Whilst I take Thomas' point about the columns being essentially the same silica based technology but with different extraction buffers I'm not quite sure of the logic of what you are proposing to do:  genomic DNA extraction columns are for purifying and recovering genomic DNA not removing it ? Unless I am mistaken by putting your DNA plus RNA through an initial genomic DNA extraction column you will probably remove some of your RNA without removing genomic DNA which is what those genomic extraction columns are designed to do.  Forgive me if I have misunderstood what you are trying to achieve 

regardkess by performing some version of your protocol I believe you would ending up losing RNA and possibly not depleting much gDNA

For an important and expensive technology like RNA seq I would advice purchasing DNAse I and either performing an on column digestion with something like Qiagen RNAse free DNAse or alternatively removing gDNA after column edition using recombinant turbo DNAse I from Ambion/  Perkin Elmer;  I have used both strategies successfully for qPCR and Micro array work

I have provided links to both products below 

DNAse ref

https://www.qiagen.com/us/shop/lab-essentials-and-accessories/rnase-free-dnase-set

https://www.thermofisher.com/order/catalog/product/AM2238

Thank you for the sugestion, Laurence. And I shoud have explained better:

My idea is adding the sample in the DNA spin-column and then retrieving the flow-through. Then, I'd add the flow-through (with the same volume of 70% ethanol) in the RNA spin-column and proceed with a normal purification of the RNeasy Mini Kit. 

I've read about the spin-columns and as far as I know, you're both right about the specificity depending on the buffers. DNA binds the silica in the presence of polyanions, that are usually present in the extraction buffers. But in the presence of >40% v/v of ethanol, the RNA binds the silica. But we still have DNA in the samples.

So my idea is adding the sample in the first silica without ethanol, so DNA would remain in the column and recovering the flow-through to the second spin-column. Then, we would proceed to purification and eluate the RNA. 

Do you think it can work?

Hi Larissa. Ok I see where you are coming from. It sounds logical. I should add that apart from using standard RNeasy columns I have also used RNEasy plus columns. These actually consist of an initial cumin step designed to remove most of the genomic DNA before adding the flow through from these columns to the RNEasy columns proper

Maybe in effect you are mimicking this set up so I would try it and see. A word of caution however. Even with these established column on column purification a however  you can lose RNA especially when starting from lower amounts of material ( usually less than 1 million cells). In the case of this RNEasy plus kit they suggest spinning for 2 min instead of the standard 30 seconds they recommend. Perhaps you could also adjust the elution time with your column ?  I should also add that for larger sample sizes - that is more than 10 million cells - this column approach to removing genomic DNA was  not always completely effective so we were then obliged to treat the final RNEasy routed material with the Ambion Turbo rDNAse already talked about

I'll add the alterations you suggested in my protocol for another pilot, prior to the real samples extraction.Thank you very much, Laurence!

How about using an RNA-specific ligase in your workflow?

https://www.neb.com/products/m0239-t4-rna-ligase-2-dsrna-ligase

This is the best solution and its so fast and easy!! https://www.thermofisher.com/order/catalog/product/AM1907

Hi Larissa

I concur with Freya if you decide after all to go down the DNAse I Digestion route; I have made ref to it myself above in one of mylinks 

The advantage of the Turbo rDNAse I is that it is just a recombinant enzyme expressed from the DNAse I ORF and thus devoid of contaminating RNAses present in many 'high grade' DNAses which thus degrade your actual RNA as well as digest your DNA. It is the only DNAse I have ever used where I have NEVER seen degradation of my RNA following treatment to remove genomic DNA. In addition, heating the RNA to inactivate most DNAses (in the presence of divalent cations) hydrolyses your RNA and the Turbo principle avoids this mode of degradation by utilising a bead based absorption/removal of the enzyme

Nevertheless, as discussed - and in the context of what you already have in house -  maybe try your column approach initially as already discussed by me; After all what you are proposing is analogous to the method used with the 'RNeasy plus kit' which utilise a column to remove most contaminating gDNA before adding the gDNA denuded material to the RNA purification proper. That said for more than 10 million cells I tend to follow this RNEasy plus procedure with a post column DNAse clean up and Like Freya I use the Turbo rDNAse I 

I have had excellent success with the Qiagen DNaseI mentioned by Laurence earlier. The on column digestion is fast and very effective for isolating high quality RNA for RNA-Seq. This approach as worked very well for me when isolating either prokaryotic or eukaryotic RNA in my RNA-Seq and RT-qPCR studies. I would worry that using the DNeasy column and buffers would allow some DNA through the wash to contaminate your RNA flow through.

Thank you all for the precious advices. Using DNase seems a very practical and logic solution, I considered it before, but it would take aprox. two months to arrive, if we order now, we don't have it here in our lab. And our situation is time sensitive. Since we already have the silica columns, we'll do a pilot extraction and then perform PCR and RT-PCR before and after the reverse transcriptase, so we'll know if there's still DNA in our samples. We won't use this approach in the real samples if there's still DNA left in the pilot samples. 

Yes, you can use columns, this is the best method to remove genomic DNA as compared to DNase. DNase can compromise the quality of RNA. RNeasy Plus Micro and Mini Kit (QIAGEN) could be a good option for you.

Can I just add a caveat to Rohits answer. In general he is right (with purified DNAses). However rDNASe I from Ambion/PE is devoid of contaminating RNases and doesn't utilise heat inactivation of the enzyme. Consequently it NEVER degrades your RNA; I can speak from 10 years of personal experience

I have isolated RNA viruses and plant viroids with the DNeasy mini columns and I have isolated bacteria and DNA viruses using the RNeasy mini columns (without on column RNase or DNase treatment). The buffers and pH do make a difference, but most likely your Promega DNA extraction kit silica columns will also bind your RNA.   

Larissa Vasconcelos could you give me a reference where I can read more about what you stated?

" I've read about the spin-columns and as far as I know, you're both right about the specificity depending on the buffers. DNA binds the silica in the presence of polyanions, that are usually present in the extraction buffers. But in the presence of >40% v/v of ethanol, the RNA binds the silica "

I can't find this information anywhere.

Thanks