Published: April 7th, 2016 Last Modified: February 21st, 2019
Working with RNA can be challenging at times. Let’s say you perform a flawless RNA prep from your (read: your PIs) model organism of choice, with the precision of a brain surgeon and the finesse of a ballerina. You store your RNA in the -80C freezer, go to sleep and dream of nice, tight qRT-PCR curves. Take out a tube a week later, run it on a gel and there’s degradation! DAMN IT! DAMN IT! DAMN IT! Goings on such as this are what lead to the neurosis and superstitions of scientists working with RNA.
Let me show you how I inadvertently reverse engineered and improved upon RNAsecure, a commercially available buffer that you can use to re-suspend your RNA pellets. Now, 10 mLs from Thermo for 175$ seems a little steep (17500$/L? LOL), considering you can make 1000 mLs for 35$!!!!!!!
RNA can degrade in solution through hydrolysis, which is enhanced by heat and high pH. Ribonucleases are a bigger problem as they are seemingly EVERYWHERE and will ruin your day. Moreover, temperature and pH can be controlled relatively easily, while tracking down the source of RNase contamination and eliminating it can be a nightmare. In all seriousness, most contamination can be avoided by just being clean, not talking with open tubes, etc etc etc. Rather than cover it all here, I will lay out all the reasonable, non-paranoid steps to take to avoid contamination in a future article.
As we all know though, even with precautions, RNase happens. Even after a good RNA prep there may be lingering traces of RNases that come along for a ride with your pellet or on the side of a tube, a spec of dust and so on. Early on I wanted to find a better way to re-suspend and store my RNA so that I could store it worry free.
Rather than reinvent the wheel, I looked to see what was out there to protect my precious RNA. Protein inhibitors of RNases are available but are too expensive for routine use, and purifying my own would be a hassle, so that was out. Thermofisher sells “THE RNA storage solution”, which they reveal is simply 1 mM Sodium Citrate buffer, pH 6.4. Bingo, a reagent that’s cheap and easy to make. The low pH of the buffer and the ability of citrate to chelate metal ions both reduce the level of RNA hydrolysis. A good start, but many RNases do not need metal ions to function, so I was still left with the problem of how to actually inactivate RNases.
I stumbled upon a patent (US 6,777,210 B1) where they describe the RNase inactivating properties of Dithiothreitol (DTT). You know, the stuff that smells like wet dog which you add to your reverse transcription reactions. After wading through a bit of legal-ese, the meat of the patent goes into detail of how you can inactivate RNases with the addition of DTT and heat! You can read the patent for your own interest, but essentially a solution of 20 mM DTT will inactivate RNase A at 60C after 10 minutes. Higher concentrations of DTT and longer incubation can inactivate higher concentrations of RNase. As well, they show that at 20 mM, DTT does not seem to have a negative impact on several types of useful enzymes (MMLV RT, T7, SP6). Interestingly the authors also use 1 mM Citrate buffer in their solutions as well. Thermo sells “RNAsecure”, a solution meant to resuspend pellets, which is then heated to 60C for 10 minutes. Sound familiar? Yep, take a quick peek at the MSDS, it’s just a solution of DTT.
So there you have it, if you want to resuspend your RNA pellets worry free, use what we can generically call “RNA Storage Buffer” (20 mM DTT and 1 mM citrate pH 6.4). You can heat the buffer before adding to the RNA pellet, after, or both. According to thermo, you can also add it to the pellet while still at 60C for extra RNase inactivating goodness.
So far I’ve had great success with this buffer, my RNA has yet to degrade even after a year of storage. The low concentration of DTT has proven to be benign to all the enzymes that I have used downstream.
To make a liter of 20 mM DTT you need 3.09 g of DTT, which from Fisher will cost you 35 bucks. The cost of the sodium citrate and water is negligible, but lets round up to 40$ for a liter. 400x less expensive than RNAsecure. Take that RNases!
Nice post. I was checking constantly this blog and I am impressed! Very helpful information specially the last part 🙂 I care for such information a lot. I was looking for this certain information for a very long time. Thank you and good luck.
I really do love this blog, I just discovered it and I’m in awe. I’ve been preparing to start RNA work in my reagent poor (chemical rich) lab but putting it off. I really cannot express how much I appreciate this!
I’m guessing you’d mention whether these need to be filtered or autoclaved, but do you pretreat the storage containers with anything? I’m horrified by the thought of sacrificing animals and not obtaining useful results.
I love you, I love this blog!
Glad some of the information was useful for you! 🙂 Good luck with your RNA work.
I don’t sterilize/autoclave the storage solution, I think that autoclaving would destroy the DTT actually. However, I do make fresh stocks every time, usually I don’t need more than a mL or two for resuspending + speccing. I have my citrate buffer aliqouted into 1 mL tubes so that I can use a fresh one each time. The 0.5M DTT I make a new 1 mL stock every 2 weeks or so, it does tend to go bad if it’s stored for more than a month. I have heard that DTT can store longer in slightly acidic conditions, but I haven’t tried it.
As for pre-treating storage containers with DTT…I’ll admit I actually did do this for a week or two, I would treat all the tubes I stored RNA in with DTT. However I abandoned this practice since it was painfully slow and I didn’t notice any improvement. Looking back on it the level of paranoia RNA work can instill can be kinda counter-productive.
Besides using filter tips, keeping stuff cold and resuspending in storage buffer, the other thing I’d recommend is that once you have your RNA resuspended, aliqout it into single serving portions and throw it in your -40 or -80 freezer. That way you take what you need for your experiment and then toss it, rather than freeze thawing your whole stock.
Beautiful, reduces disulfide. This won’t be permanent (RNAses will re-fold if the DTT oxidizes) but a great idea. Regarding storage of DTT, it fares better in acidic conditions because those are generally reducing conditions (for biological compounds). You can find some old papers that show that DTT decomposes very quickly as the pH approaches 8.
For that reason, TCEP would probably work even better since 1) it is much less sensitive to pH 2) it does not react with thiol-reacting agents. The downside of TCEP is that it is incompatible with phosphate buffer, but those are typically not used for nucleic acid work anyways.
You’re absolutely correct, I’ll change the wording in the text. I pulled the “irreversible” tidbit from the patent (https://patents.google.com/patent/US6777210B1/en), but they were not testing their own work, just defining the term. Thank you!
I’ll take a look at TCEP, I’m always looking to improve my RNA game. Allegedly you can add a small amount of bleach to your agarose gels to reduce RNase activity, and to denature the RNA, been wanting to try that out as well (https://www.ncbi.nlm.nih.gov/pubmed/22222980).
The RNAsecure claims” A nonenzymatic reagent that irreversibly inactivates RNases in solutions that..”.. How is it irreversible if its only DTT?
Simply because S-S bond formation in RNase requires glutathione, which is not present in the storage buffer. RNase activity inhibition by DTT is therefore irreversible and there is no point of replacing DTT by TCEP in storage buffer:
https://pubmed.ncbi.nlm.nih.gov/15604666/
There may be a benefit in replacing DTT with TCEP in lysis buffer though.
One vote for using 10 mM TCEP versus DTT for stabilization of RNA.
https://www.ncbi.nlm.nih.gov/pubmed/14715294
Hi there,
Just a thought. I recently discovered that it does not take much DTT (1mM or greater) to inhibit the activity of DNase. So if you do DNase treatment after resuspending your RNA in a buffer with DTT, maybe your DNase treatment will not be as efficient? Like I said, it’s a thought, though I am not sure how much of an issue it is.