Dear All, I posted the following question on EvolDir about a week ago: 'Dear Colleagues, I am attempting to extract DNA from RNAlater stored tissue samples for downstream DNA methylation sequencing (MS-AFLP, MeDIP-seq). I have tried several different extraction protocols: Qiagen DNeasy blood & tissue, Promega Wizard, Phenol/Chloroform and a spooling protocol from Thermo-Fisher specifically intended for this type of DNA extraction. All of these extraction protocols I followed-up with isopropanol precipitation to clean the extracts further. Whilst I successfully extracted some relatively high quality DNA (when visualized on a gel), yields were lower than expected. More worryingly the 260/230 ratios were low (<1.2) suggesting that a lot of salts were being co-extracted alongside DNA. My main concern here is that these salts could impede enzyme action during restriction-ligation or PCR leading to problems with reproducibility. Has anyone had some success extracting DNA from RNAlater stored tissue and would be willing to share some tips/advice on how to carry these extractions out successfully and cleanly?' I received some excellent replies and also several requests to post the replies. Please find below the collated responses to my query in rough chronological order. Many thanks to all those who took the time to help me with my problem Best wishes Alan (alan.hudson@bristol.ac.uk) ----- Hi Alan, I was asked this question recently by a colleague. As was the case then, I have not actually tried to do this myself, but came across the following when looking for Chelex-based extraction protocols. https://www.researchgate.net/post/Is_that_possible_to_extract_DNA_using_Chelex_from_RNALater_preserved_samples Maybe this is of help? Or maybe is just another version of what you’ve already tried. Anyways, good luck! David Garfield ----- Hi Alan, WE had some issues before with RNAlater-preserved samples. What we did was we washed our samples with 1x PBS to get rid of the salts. Our DNA quality and PCR amplification was better afterwards. Best, Marilou Sison-Mangus ----- Dear Alan, We used a kit that should be analogous to the Qiagen kit (silica based column), but we did not purify it further (just followed the instructions of the kit). You can see our results in the attached paper (gel in Figure 3- NAP turns out to be the same as RNA later). Good luck! Jennifer Leonard ----- Dear Alan, we have had a very good experience rinsing RNA-Later fixed samples in PBS (1X) before homogenization. We do this for RNA extraction. If we use CTAB instead of Trizol, we always recover very high quality DNA from RNA-Later fixed specimens and clean the DNA using the Zymo Duet kit (this usually cause the DNA to break, but we are only interested in RNA...). How long, this we never tested. We ussually transfer the sample from RNA-Later into PBS and wait for, say, no longer than 5 mins before transfering the sample to lysis buffer. Long ago, I read this PBS wash step helps getting better RIN values, likely due to salt removal. Maybe it does the trick. cheers Sergio Vargas ----- Dear Dr. Hudson, I have extracted DNA from tissues stored in RNAlater using the classic Phenol/Choroform/Isoamyl alcohol (PCI 25:24:1) protocol without any modification. After extraction, there were huge white precipitates in the tubes as I guess happens to you. Therefore, I performed an extra step of cleaning-up with magnetic beads (AMPure beads). After this clean-up I have used these samples for RRBS and also specific PCRs after bisulfite conversion and everything worked fine. If you need further details on the clean-up step, please let me know. Best regards, Dafni Anastasiadi ----- Hi Alan, re your evoldir question - Isopropanol tends to leave more salts. I would use >95% ethanol to precipitate then wash pellet with 70% ethanol to remove even more salts. Personally I've never had a problem with RNAlater preserved tissue. Though I've only ever PCRed it, nothing fancier. Secondly I've never been a big fan of 260:280 as a measure of quality. DNA not cleaned of salts can both PCR and restrict just fine. Though you need to check for yourself if it is in your experiment. Cheers Dave Lunt ----- Hey Dr. Hudson, We extract DNA from microbes on filters stored in RNAlater (or a homemade equivalent, anyway) all the time. For us, the key is to rinse the filters thoroughly before starting the extractions. I don't know how easy this would be for you if you have tissue samples, but we just put the filters back in our filtering apparatus and run a few hundred milliliters of ultrapure H2O through... Hope you get your problem solved. Good luck! Mattias Johansson ----- Hi Alan, I have had great success extracting DNA from tissues stored in RNAlater. I use a Qiagen DNeasy extraction kit. What i do first is, rinse the tissues in DNA grade water three times. Then I soak them in water for an hour then proceed with the normal extraction protocol. I have used these extraction in sequence capture methods with no problems and with high yield of quality DNA. I hope this this helps. All the best, Perry Wood ----- Hi Alan, This is in response to your RNAlater question on evoldir. We commonly use RNAlater for stabilization of DNA in tissue, buccal swabs, and fecal samples, and perform a TE soak prior to DNA extraction to remove salts, as did Michaud et al 2011. Here's our protocol for bat buccal swabs. Step 5 is relevant to your tissues. 1. Buccal swabs should be collected in 500 ul of RNAlater, then frozen at -20 or -80 °C. 2. Extract DNA in batches of no more than 20 tubes. 3. Defrost tubes, then spin to pellet cells (10 min at 7500 rpm). 4. Gently remove RNAlater, so as not to disturb pellet. Swab will still contain RNAlater, but that’s okay. 5. Perform a 1 " 2 hour soak in 1 x TE (500 ul, vortex; following Michaud et al. 2011, and my optimization results). 6. Spin again (10 min at 7500 rpm) and gently remove TE. 7. Begin Qiagen DNEasy Blood & Tissue Kit DNA extraction using Animal Tissue Spin-Column Protocol (page 28), which involves an overnight incubation (with swab still in tube). At final step, elute in 100 ul AE buffer, and also do a 2nd 100 ul “B” elution, which can be stored in a separate box in -80 °C in case we need it later. 8. Store all DNA extractions in -80 °C. I hope this helps. Good luck! Best, Faith Marguerite Walker ----- Hi Alan, We have recently tried doing just this. Try bathing the tissue in TE buffer or molecular grade water for a couple of hours prior to extraction. This may help remove salts from the tissue. All the best. Erica V. Todd ----- Hi Alan, My lab used salt extraction protocols (for gDNA) so I don't know how much help this will be. The use of isopropanol seems off for the cleanup stages as we use it as a precipitation step. I would probably try cleaning up your extracts by precipitation then 70% ethanol washes to remove the salts. So long as the ethanol is pure or not denatured with benzene it should help. Stephen Rice ----- Dear Alan Yes, RNAlater has a lot of salts that are indeed co-extracted and precipitated. Although I have never tried to clean up RNAlater DNA isolates, we were successful in cleaning a lot of low quality gDNA with the NucleoSpin gDNA clean-up kit (http://www.mn-net.com/tabid/11745/default.aspx). Otherwise I would recommend precipitation with 2x volume of -20°C ethanol overnight, instead of the isopropanol. The latter is indeed a much faster precipitation, but also much more dramatic and other components normally co-precipitate. Best wishes Ovidiu Paun ----- Using the kit of Nucleospin gDNA by MacheryNagel and using 50 µL of its elution buffer improved my 260/230 ratio to exact 2.0-2.2 values Cheers Britta Meyer ----- Dear Alan, I tried for a long time to extract DNA from a very tricky invertebrate (Sabellaria alveolata) using samples stored in RNA later. I ended up using a nasty guanidine thiocyanate extraction and this gave me reasonable (although not great) quantity and quality of DNA. One trick, that you may already be doing, is to rinse your samples with PBS after removing them from the RNA later, before beginning the DNA extraction. This gets rid of any excess RNA later and the salt crystals that can sometimes form on the samples when you use RNAlater to store them. I hope this helps! I am very interested to hear what other suggestions people came back to you with and it would be great if you could share them. Best wishes, Anna Muir ----- ah15824@bristol.ac.uk