Published: May 8th, 2016 Last Modified: June 16th, 2017
Extracting RNA from tissue is one of the most critical procedures in RNA work, considering that un-degraded RNA is the starting point for all your downstream experiments. Usually, this means taking your tissue, grinding it up in buffer and going through your standard RNA purification procedure to completion where you can breathe easy (not over the tubes!) ONLY after your RNA is safely re-suspended in RNA storage buffer, aliqouted and frozen.
Sometimes extracting RNA to completion isn’t practical, whether it’s because you’ve got 20 different samples, you’re tired and it’s 2 AM or you’re collecting samples in the middle of a jungle (Perhaps all three?). Times like these call for RNAlater Stabilization Solution, a Thermofisher product, which allows you to immerse your tissue to process later without fear of RNA degradation. Nice right? Only problem is that it costs between 800-1200$/L depending on if you buy in bulk. Turns out you can make your own for 50$/L!
Before re-inventing the wheel and spouting off about what a special reverse engineering snowflake I am it’s always good to do a quick search to see if people have already tried to make their own RNAlater. Glad I did, because it seems it’s been done to death already. The patent essentially spells out how to make the stuff:
“In a beaker, combine 40 ml 0.5 M EDTA, 25 ml 1M Sodium Citrate, 700 gm Ammonium Sulfate and 935 ml of sterile distilled water, stir on a hot plate stirrer on low heat until the Ammonium Sulfate is completely dissolved. Allow to cool, adjust the pH of the solution to pH 5.2 using 1M H2SO4. Transfer to a screw top bottle and store either at room temperature or refrigerated.”
Not much I can add here except a cost breakdown. All prices are the cheapest I could find to make 1 liter, the supplier is Fisher, not including shipping.
- Ammonium Sulfate – 10 kgs for 532$ = 0.053$/gram * 700 grams = 37.24$
- EDTA Disodium Dihydrate – 100 grams for 41$ = 0.41$/gram * 7.44 grams = 3.o5$
- Sodium Citrate trisodium salt, dihydrate – 1 kg for 66.54$ = 0.066$/gram * 7.35 grams = 0.49$
- Distilled water – Negligible
- 1M Sulfuric Acid to pH to 5.2 – Negligible
- NaOH to pH EDTA to pH 8.0 – Negligible
So, less than 50$/L assuming you have water, sulfuric acid and NaOH? 20 times less than Thermofisher, not shabby at all.
I’m wondering – and I would love to hear especially from those scientists who actually tried to compare some of the RNA preservants and then they checked the quality of the isolated RNA – how do they compare?
I have tried
Hi I have compared RNA later reciepe with Qiagen RNA protector for storage of heart tissues and got excellent results in RNA gel even after 1 week of storage @-80…It works as good as commercialy available one
Glad to hear it, that’s awesome 🙂
Did u had any trouble preparing it? Because mine would precipitate all the time. I got some pretty crystal forms after cooling that I suppose I shouldn’t have got. I ended up filtering to get rid of them.
Initially we had trouble in dissolving …but it got resolved and we did not get any crystals after cooling.
I also had a similar situation occurring, it’s the ammonium sulfate precipitating out. Did you adjust the pH to 5.2? That helps some, however others who have tried the recipe still get crystals. I think filtering is a good solution, would be equivalent to adding less ammonium sulfate in the first place. I think leaving the crystals in there wouldn’t hurt either. Other tissue preservation media has been proposed: https://www.nature.com/nmeth/journal/v11/n5/full/nmeth.2910.html#methods
“RPHB: 300 mM NaCl, 30 mM sodium citrate, 2.1 M ammonium sulfate, 10 mM EDTA, 1 mg/ml Escherichia coli tRNA, 500 μg/ml BSA, 25% (40%, v/v) formamide for 20- (30-)nt probe library, pH 5.2; wash buffer: 25% (40%, v/v) formamide for 20- (30-)nt probe library, ”
They get away with “only” 2.1M ammonium sulfate, the DIY RNAlater is something like 5M, so I don’t think filtering out the crystals would hurt any.
I adjusted the pH at the end only.
I had previously used a saturated solution of NaCl to preserve DNA and mostly worked. So, I thought that probably a saturated solution of ammonium sulphate should work for RNA, so I just filtered the excess.
Knowing that they used 2.1M makes me more confident.
Thanks a lot, guys!
I think the DIY RNAlater is not 5M. The protocol saying: 700g + ~ 935 ml water. This will not make 1 liter final solution, which will be over 1300 ml. Same method is to make 100% saturate ammonium sulfate solution at 0C, which should be around 3.8 M.
Hello again,
I have stored samples in RNAlater for RNA extraction, but now I am interested in extract DNA from those.
Since I’m back to work in a developing country, saving money is a must, so I have to work with ctab-based protocols. The problem is that the pellet is too big to be true (twice as the size of a fresh sample), so I believe that the salt from RNAlater is precipitating too. The regular ethanol cleaning step is not helping.
Has anyone a recipe on how to remove excessive salt from the eluted DNA or a protocol to use with RNAlater for DNA extraction?
I thought on using the NucleoSpin Gel and PCR Clean-up from MN, but I am running out of the NT3 buffer. What do you think?
cheers,
Hello! I found that even with TRIzol reagent for extracting RNA I was getting extra salt coming out in the final pellet. Without modifying the protocol, you need to get rid of as much of the RNAlater as possible, like blotting it off with filter paper and/or washing once with ddH2O before extraction.
One option, don’t know if it’s ideal cost wise, but sephedex G50 resin ultra fine grade will desalt nucleic acids pretty good, you need the little filter columns to hold the resin, enzymax has the cheapest http://www.enzymax.net/columns/filter_column.htm
As for silica spin kits, MN is among the most expensive kits (mmm German made kits). The cheapest standalone kits I’ve found yet are http://www.enzymax.net/columns/DNA_isolation.htm, and they provide buffer recipes for the various cleanup operations you can perform with them. I bought their 100 pack of spin kits so I could tell what buffers they were suggesting to use, and to compare them to what I’ve been able to reverse engineer…turns out they’re suggesting using Qiagen buffers with their spin kits. Pretty cheeky, eh? Looks like they reverse engineered them independently 😀 Here is their recipe, mine doesn’t use Tris and uses isopropanol: Qiagen Buffer (Enzymax variant) PB :5 M Gu-HCl, 20 mM Tris-HCl pH 6.6, 30% ethanol, add 5 volumes to 1 volume of PCR/enzymatic reaction, load onto column. This should work on your leftover MN columns too.
Silica spin kits could work for desalting, give it a go! I guess keep in mind the maximum amount of DNA one of those things can bind is like, 20 ug under ideal conditions.
Hi Pipette Jockey!
Thank you so much for your help.
I am going to try with the kits I have.
I brought from Germany some leftovers from my post-doc, including 1 kit for gel and pcr purification from MN and another for plasmid from Qiagen.
All the best.
Do you know Trizol-Ls Composition?
Is this solution autoclaveable?