RNA extraction and DNase treatment

[rubyryan]

Hi there,
I am planning to carry out a small RNA sequencing project and would like some advice on the RNA extraction/ DNase treatment protocols. One of my plans is to use the MirVana Paris kit followed by Ambion DNase treatment (either Turbo or DNA free kits). Anyone tried these methods? After using the Ambion kits is it necessary to filter purify to remove the DNA removal reagent or is simple pipetting the RNA enough? I really want to avoid any contamination.

My other choice is to use the Norgen RNA extraction kits and do an on-column digestion with Invitrogen. It would avoid any post-RNA extraction procedures but may give a lower yield.
I will perform the small RNA sequencing on the Illumina HiSeq

Any advice/ knowledge would be gratefully appreciated.

[lmolokin]

In my experience on-column DNase treatment is never as efficient at removing gDNA as in-solution DNase treatment. Anytime an RNA extraction protocol has an on-column DNase treatment step, I skip it, and follow up the extraction with Turbo DNase. With the Turbo kit you don't have to worry about purifying your RNA afterwards as the final step of the protocol uses an inactivation reagent to remove the enzyme and divalent cations from the RNA.

[rubyryan]

Many thanks lmolokin for your comments. IF you have any advice on concentrating the RNA with minimal loss please let me know as my RNA will need to be concentrated. Thanking you.

[lmolokin]

Happy to help.

I prefer to use vacuum centrifugation to concentrate my RNA, however, when doing so try to avoid spinning the RNA under vacuum for prolonged periods of time. I also typically never concentrate a volume greater than 40uL as I've had issues with RNA loss and degradation with larger volumes. Aliquot your sample into multiple fractions if you have to--this cuts the spin time down as well.

With my equipment, it takes about 10-12 minutes to concentrate a 40uL volume down to about 15-20uL.

[protist]

I would recommend RNA Zymo Clean and concentrate columns (5ug) we use them instead of precipitations in our library protocols.

[rubyryan]

Thanks for that! Further to this, once I have obtained the ideal total RNA concentration, (200 ng/ul) I am going to try and sequence all of the small RNA (ie microRNA and piwiRNA) using the Illumina TruSeq small RNA library preparation and then sequence on the HiSeq Illumina. Is this feasible or is it better to focus on one family of small RNA?
Thank you

[chadn737]

I have used Turbo DNase followed by cleanup on Qiagen columns. The Turbo DNase kit does provide an inactivation agent that is supposed to remove the enzyme, but time and again I find that the inactivation agent fails to adequately remove the enzyme (I have been using it for ~ 5 years now). For more routine uses, I'll use the inactivation agent, but for my more valuable samples, I always follow up with cleanup on a column.

[pmiguel]

Originally Posted by rubyryan

Thanks for that! Further to this, once I have obtained the ideal total RNA concentration, (200 ng/ul) I am going to try and sequence all of the small RNA (ie microRNA and piwiRNA) using the Illumina TruSeq small RNA library preparation and then sequence on the HiSeq Illumina. Is this feasible or is it better to focus on one family of small RNA?
Thank you

The Zymo columns mentioned up-thread seem to work well.

If you are using the Illumina TruSeq small RNA prep kit, then there is no need to isolate small RNA. It is designed for 1 ug of total RNA substrate. Even the DNAse treatment may not be needed -- it is recommended so that the amount of total RNA can be accurately estimated. But if you have a fluorimeter, like a Qbit, you could just use an RNA-specific fluor to determine your RNA concentration.

We have sequenced a larger "window" of small RNA sizes. Ended up running them in a 100 nt run, because no other 50 nt samples were in the queue. Looks like we got sequence of both miRNA and some other (larger) RNAs. Analysis isn't back yet, but I guess it worked.

--
Phillip

[rubyryan]

Many thanks for your comments. Very helpful.

[Johan de Bondt]

Originally Posted by chadn737

I have used Turbo DNase followed by cleanup on Qiagen columns. The Turbo DNase kit does provide an inactivation agent that is supposed to remove the enzyme, but time and again I find that the inactivation agent fails to adequately remove the enzyme (I have been using it for ~ 5 years now). For more routine uses, I'll use the inactivation agent, but for my more valuable samples, I always follow up with cleanup on a column.

If someone would be interested, we have developed an IP protected magnetic bead with active DNase on the surface. Perhaps an elegant manner of removing the DNA without loosing sample or diluting it! If anyone would be interested in this feel free to send me a mail.

[josdegraaf]

Originally Posted by Johan de Bondt

If someone would be interested, we have developed an IP protected magnetic bead with active DNase on the surface. Perhaps an elegant manner of removing the DNA without loosing sample or diluting it! If anyone would be interested in this feel free to send me a mail.

where to send the email?

[pmiguel]

Originally Posted by Johan de Bondt

If someone would be interested, we have developed an IP protected magnetic bead with active DNase on the surface. Perhaps an elegant manner of removing the DNA without loosing sample or diluting it! If anyone would be interested in this feel free to send me a mail.

Problem would be the DNA would degrade, but be left in solution with the RNA. So you end up having to do a size fractionation anyway to remove it.

I actually think the Zymo DNA-free RNA purification is the way to go for this. Your RNA prep is done and fairly clean. Hit it with a DNAse followed by a column clean-up to both remove the degraded DNA as well as the DNAse. As a bonus you also remove any other residual proteins or contaminants still remaining in your prep.

--
Phillip

[Papaveraceae]

Has anyone used RNAzol, from Molecular Research Center, they make the claim that DNAse is not required after extraction. See the link: http://www.mrcgene.com/rnazol.htm

Dave

[Papaveraceae]

Originally Posted by Johan de Bondt

If someone would be interested, we have developed an IP protected magnetic bead with active DNase on the surface. Perhaps an elegant manner of removing the DNA without loosing sample or diluting it! If anyone would be interested in this feel free to send me a mail.

Johan,

I am interested in your IP protected magnetic bead with active DNase on the surface. Is the information available online if not please let me know more about it via email.

Thanks,

Dave

[ehdezsanabria]

Originally Posted by chadn737

I have used Turbo DNase followed by cleanup on Qiagen columns. The Turbo DNase kit does provide an inactivation agent that is supposed to remove the enzyme, but time and again I find that the inactivation agent fails to adequately remove the enzyme (I have been using it for ~ 5 years now). For more routine uses, I'll use the inactivation agent, but for my more valuable samples, I always follow up with cleanup on a column.

Hi chadn 737,
We did the DNA removal using the Turbo DNase from Ambionbut used phenol:chloroform to inactivate the enzyme. Do you think this step is necessary or else, can you proceed directly to using the RNA clean-up using a kit (Qiagen)?

[MatthewHaas]

Originally Posted by chadn737

I have used Turbo DNase followed by cleanup on Qiagen columns. The Turbo DNase kit does provide an inactivation agent that is supposed to remove the enzyme, but time and again I find that the inactivation agent fails to adequately remove the enzyme (I have been using it for ~ 5 years now). For more routine uses, I'll use the inactivation agent, but for my more valuable samples, I always follow up with cleanup on a column.

This thread is rather old, so I'm not sure if I'll be able to get a response, but here goes...

When you do the column purification to remove the DNase enzyme, do you add buffers (which buffer/what volume?) to your RNA sample before passing it though the column and proceed with the same protocol you used initially to extract the RNA? (With Qiagen Mini kit: RLT, RW1, RPE.) I need to remove some DNA from my RNA samples and I'm a bit concerned that you said the inactivation agent isn't completely removed. I want to make sure the cDNA created from my RNA libraries isn't automatically digested. Thanks for your input.

[DStephens@NuGEN]

Hi MatthewHaas,

The Qiagen RNAeasy kits include separate protocols for RNA Clean-up after DNase treatment. All you need to do is bring your sample volume up to 100 uL with nuclease-free water, add RLT and EtOH as directed, and add to the column. See p. 54 of the RNeasy Mini Handbook here: https://www.qiagen.com/us/resources/...a33e24&lang=en

[Amar]

That's really interesting thanks DStephens@NuGEN. I never knew the RNAeasy columns can be used for clean up.

MatthewHaas: The turbo DNase kit is fantastic and in my experience the inactivation buffer works great. Just be sure to stay well away from the inactivation pellet. I have also been told that DNAse is extremely fragile and sensitive to heat. cDNA synthesis includes a heat-inactivation step, which should hopefully degrade DNAse. But as DStephens@NuGEN said, if the samples are precious then perform a cleanup step.

Good luck!

[kumard]

Originally Posted by Papaveraceae

Has anyone used RNAzol, from Molecular Research Center, they make the claim that DNAse is not required after extraction. See the link: http://www.mrcgene.com/rnazol.htm

Dave

Its a very old thread, but let me add that I am using RNAZol RT and I see DNA contamination even after following their protocol. I am struggling with this problem. I talked to their tech support and they informed me to do DNAse I treatment. The advantages that RNAZol RT have is that you can use water for the phase separation and the smallRNA fraction profile is better (on small RNA sizing on Agilent bioanalyzer) than Trizol (with RNA of RIN 9). I hope it will help someone.