Question

Degradation of DNA during RNase A treatment of previously extracted DNA

I wonder if anyone here has had any problems of treating already extracted DNA samples with RNase A, and if you have identified the problem or found a solution. I do know that it is preferred to add the RNase A during extraction, but we have samples (generally extracted with a Phenol-Chloroform protocol) for which there is no original blood/tissue left, and which need to be RNase treated. Thus, we have used Qiagen's RNase A and followed the protocol, which in short is:

Add 1 μl of 10μg/ml RNase A to your sample. Incubate at 37C for 30 minutes.
Add 1/10th volume of 3M NaAc and 2 volumes of ice cold 95% ethanol.
Let rest in freezer for 30 minutes. Spin for 10 minutes at 13.000 rpm.
Remove the ethanol. Add 100 μl ice cold 70% ethanol. Spin for 10
minutes at 13.000 rpm. Remove ethanol. Dry samples in centrifuge.
Resuspend DNA in 1X TE buffer.)

However, for many (most) samples, what was previously DNA of high concentration (and high quality) turns after RNase treatment into very low concentration. I.e. we lose the DNA in the process.

This is not owing to that the original samples would have consisted of mainly RNA; on a gel they would produce a strong band of high molecular weight DNA and little or no smear of shorter fragments.

I'd be grateful for any help on this, since we would want to be able to confidently RNase treat our samples to get rid of RNA, but not risk losing much DNA.

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  • Berthold Heinze · Federal Research and Training Centre for Forests, Natural Hazards and Landscape
    DNase contamination in any of your reagents (including the RNAase)? Or insufficient precipitation process? Lost pellet in the "dry samples in centrifuge" step?
  • Martin Stervander · Lund University
    Hi Berthold,

    Thanks for the suggestions! The RNase A is supposed to be endonuclease-free (guaranteed by the manufacturer), and the remaning reagents are used during regular DNA extraction, so it's not apparent where the contamination would be. Also, while the vacuum centrifuge step has potential risk of losing pellets, this is usually not a problem in any other context. If my memory serves me, I think we once (long ago) saw the loss of DNA already after incubation with RNase A. I will ask my colleagues (who are in the lab while I'm on computer duty at the moment) to quantify already after the initial incubation.
  • Berthold Heinze · Federal Research and Training Centre for Forests, Natural Hazards and Landscape
    Martin,

    btw, a DNAse contamination test would be rather easy to do with any of the reagents - just incubate a bit of sound DNA with the reagents, and do a gel with that DNA before/after incubation.
    Many years ago, I tried to isolate DNA from birch pollen - very unsuccessfully. In the end I found out that the pollen, once in contact with water, degrades added DNA very efficiently ...
    So if any of you ever run out of DNase in the lab ... just use birch pollen :-)
  • Martin Stervander · Lund University
    Haha, that's a great and cheap substitute... Who would have thought birch pollen had such superpowers. Will suggest the simple DNase contamination test. Thanks again!
  • Been there, seen that.
    Buy a fresh stock of Qiagen RNAse.
    Good luck.
  • Bastian Herzog · University of Prince Edward Island
    Hi, had the same problem of low DNA yield. Precipitation was, in my case not efficient enough, so I extended the "Let rest in freezer for 30min" step to "Let rest over night". That increased yield. Good luck!
  • Cynthe Sims · VantagePoint Laboratory Partners
    DNAse co-purifies with RNAse, many commercially available preps contain traces of DNAse. Fortunately RNAse is extremely thermostable. You can boil yor RNAse for 10 minutes to denature the DNAse and then centrifuge to pellet out any contaminants.
  • Mrs. Smitta Tripathi · University of Delhi
    Hi........I would like to know more details such as thermal stability of the DNA of your interest at 37 degrees for 30minutes because if the DNA is getting denatured while/after incubation, it's each strand is now susceptible to RNAse degradation..............have you checked the above situation by decreasing incubation time (here again you will face problem with RNAse activity that may decline)
  • Jetty Ramadevi · University College Dublin
    May I know what is your plan to do with that DNA?

    If you are going to run a PCR, you hardly get any trouble with that RNAse as it is going to degrade when u do PCR with initial degradation step itself i.e. 94 C.

    By the way what was your sample volume, if you have enough carry again phenol:chloroform extract your DNA after the RNase digestion very carefully.

    or

    if your DNA is partly degraded I do not think it gives any serious problem to your future work.

    I feel even you have not given any treatment after treating with RNAse it works (check the manufacture's instruction about the activity of RNAse).

    Cheers!!!
  • Gaurav Sharma · Institute of Microbial Technology
    Hey martin,
    I have also faced this problem many times.
    I would suggest to do RNase treatment before PCI-CI step, as the more times you would do ethanol precipitation, the DNA loss would definitely occur.
    Besides this in your case, it is sure that your eppendorfs or any other solution (Sodium acetate or any other) is causing problem. Try to autoclave eppendorf and make new solutions. Although loss would be there, but it can be decreased at some extent.
    I generally go with 1st step. Do RNase treatment before PCI-CI step.
    Good luck.
  • Siva Sivaramakrishnan · Acharya N G Ranga Agricultural University
    DNase contamination is the obvious reason for your reduced yield. You can either increase the temp to 60oC when the Rnase will be still active but not the DNase or you can include a small concentration of EDTA which will scanvenge the divalent ions helping the DNase.
  • Berthold Heinze · Federal Research and Training Centre for Forests, Natural Hazards and Landscape
    May I point the esteemed readership to a really old article I wrote way back then:

    Heinze, B. (1994). RAPD reactions from crude plant DNA. Molecular biotechnology, 1(3), 307-310. Abstract: As opposed to standard polymerase chain reaction (PCR) using specific primers, genome analysis involving short random primers, for example RAPD, may yield inconsistent results if crude plant DNA preparations are used as the template. When RNase A, a thermostable enzyme, was added to such reactions, highly repeatable banding patterns were obtained from crude plant DNA, thus speeding up analyses substantially.

    Sorry for the self-ad, but some of the findings there seem to still be relevant here ...
  • Martin Stervander · Lund University
    Hi all and thanks for the comments. I'll try to reply to some of them, and also summarize experiences that others have share on an email list.

    Andrija, this issue is not confined to a particular batch of the Qiagen RNase A (the stock was freshly ordered), but has happened in different batches. This has also been confirmed now by several other users, and it seems like the Qiagen RNase A generally has this effect on already extracted DNA. I would therefore avoid using it. Whether this is because it isn't truly DNase-free or not, I am not sure, but I guess it may be the reason. Other users, which have also struggled with failed DNA samples after Qiagen RNase A treatment have later successfully treated their (already extracted) DNA with Thermo's DNase-free RNase A.

    Thanks for the alternative suggestions to inhibit the activity of DNase (especially Siva; increased temperature and/or addition of EDTA during enzyme digestion) or lower the DNA loss by longer ethanol precipitation (Bastian)! Or degrading DNase by boiling the RNase A (Cynthe).

    As stated in the original post, it is of course better to perform the RNase treatment on digested samples before the actual DNA extraction (Phenol-Chloroform in our case), but this is not an option for these already extracted samples for which no original blood sample is available.

    Jetty, the DNA will be used for RAD sequencing, for which optimal yield of even representation between individuals among RAD tags high molecular weight DNA is preferred. I agree that for regular PCR's for Sanger sequencing this would not be an issue at all. Performing another Ph-Chl extraction of the samples after incubating the RNase A might be an option. Berthold and Jetty, the RAD sequencing is (as you may know) based on a first restriction enzyme digestion, so this publication probably has some bearing!

    Smitta, it is high molecular weight DNA, so it definitely wouldn't denature at 37 degrees!

    Cheers,
    Martin
  • Hi Martin, spot on! You can always boil it (Anfinsen got Nobel Prize because of RNAse stability). This should denature and remove DNAse, but still some other endonucleases may be heat resistant. Personally, I never use RNAse in the last step of DNA extraction and it always needs to be removed in subsequent steps.
    Cheers
    Andrija
  • Jetty Ramadevi · University College Dublin
    Anyway, though your DNA is partly degraded I hope it should certainly possesses some of RAD tags that you are going to flank (no idea what restriction enzymes are you going to use). Wish your RAD tagging should go smoothly.
    Good Luck!!
  • Farid Elasmer · Ain Shams University
    From what I can see the only explaination is that some how you are contaminating your samples with dnase
  • Jessica Prebble · Massey University
    Hi Martin,

    Thanks for sharing your question, I am having exactly the same problem as you, and Qiagen RNAse is making my DNA from precious samples disappear. Just wondering what solution you came up with? Did you end up using a different brand of RNAse with success, or boil the RNA, or change the protocol... or something else? I am finding it very confusing as when we use the exact same RNAse during our extraction protocol it works fine. I'm off to try boiling some now...

    Thanks,
    Jessie
  • Martin Stervander · Lund University
    Hi Jessica,

    Sorry for not replying promptly. I'll give you two replies: Firstly, eventually I realized that for the RAD sequencing it was not really necessary to treat the samples with RNase A. In the end, this is what we ended up doing - avoiding RNase treatment. As long as you quantify the samples accurately (with a DNA specific assay, as with a Qubit), it doesn't really matter that there is also some RNA present.

    Secondly, if you actually do need to treat your samples, and if they have been extracted previously, there's no idea trying with Qiagen's RNase A. However, from other people that responded to a post on an email list, I concluded that they have successfully treated previously extracted samples with DNase-free RNase A from Roche and Thermo, respectively. I think that these protocols suggest an extra precipitation following the treatment, in order to get rid of remaining enzyme.

    I hope that this information will be helpful, and I do hope it doesn't arrive too late...

    Best,
    Martin
  • Jessica Prebble · Massey University
    Hi Martin,

    Thanks for your reply. I ended up using a work-around also - I was attempting to get rid of RNA in order to pool multiple DNA extractions to send off for an illumina miseq run so I did need to get rid of the RNA - but ended up using a repli-g kit on one of the good (i.e. no RNA) samples instead... which worked beautifully...

    Interestingly I did actually manage to get rid of RNA and keep the DNA using the Qiagen RNAse A by essentially re-extracting the DNA extractions that contained RNA using the Qiagen DNeasy kits, but I lost too much DNA along the way for it to be useful to me.

    Best wishes,
    Jessie

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