In good conditions, you can store RNA for an extended period. If RNA is in a stable buffer with RNase inhibitors, it can even stay at room temperature for multiple days without much degradation.

But as a general rule, I would recommend to store RNA at least in -80°C and most importantly, monitor the quality. There are multiple ways: automated electrophoresis systems (Agilent's Bioanalyzer, Bio-Rad's Experion, Calliper's Labchip...), or other methods, 5'-3' assay (in case you want to use qPCR afterwards).

So in conclusion, don't believe too much of the stories and the myths that are going about on RNA. Just check the quality and see for yourself what conditions are best in your lab.

By the way, low quality RNA does not always meen that your samples will be useless. If you are working with archieval parrafin embeded material for example, than it is better to get at least some data (with higher variation) than nothing. Just make sure that you only compare those samples with equaly degraded RNA.

The best way to get rid of this problem is to use the RNA as soon as possible....withing 12 hours, or u could rely on nitrogen for preservation but as liquid nitrogen transfer and use is banned...we r left with only option of using the RNA as soon as possible..................

In good conditions, you can store RNA for an extended period. If RNA is in a stable buffer with RNase inhibitors, it can even stay at room temperature for multiple days without much degradation.

But as a general rule, I would recommend to store RNA at least in -80°C and most importantly, monitor the quality. There are multiple ways: automated electrophoresis systems (Agilent's Bioanalyzer, Bio-Rad's Experion, Calliper's Labchip...), or other methods, 5'-3' assay (in case you want to use qPCR afterwards).

So in conclusion, don't believe too much of the stories and the myths that are going about on RNA. Just check the quality and see for yourself what conditions are best in your lab.

By the way, low quality RNA does not always meen that your samples will be useless. If you are working with archieval parrafin embeded material for example, than it is better to get at least some data (with higher variation) than nothing. Just make sure that you only compare those samples with equaly degraded RNA.

Additionally you can use Low Binding Tubes or Plates (f.g. Eppendorf Tubes): http://www.eppendorf.com/int/img/lobind/Flyer_LoBindTubes_GB_V10.pdf

That's maybe also one thing to keep in mind...?!

Best, Nadine

The answer to your problem will depend on the final use of the RNA products. For example, if the RNA represents RNA transcripts that you have prepared as a positive control for a RT-PCR assay I would suggest you add some carrier RNA (non specific rRNA or tRNA) to minimise degradation of the target RNA transcript and then store at -80C in small aliquots.

I would like to thank all of you for your helpful suggestions.

Under ethanol and 3M NaAc, -80C. I've worked (RT-PCR) with RNA (ssRNA virus) in three years after keeping in this condition.

Store isolated RNA in Formazol at -80 C. It remains stable for years. It will need to be reprecipiated by ethanol to remove Formazol which inhibits RT, I believe in the presence of EDTA. RNA in Formazol can also be run on denaturing gels directly after suitable additions.

RNA quality from culture cells normally is better than this from tissue, you can check it running on denaturing gels in order to see RNA ribosomal bands. I resuspend RNA in nuclease free water.

We work mainly with RNA in our lab and I store my RNA in nuclease-free water at -80. A couple of times thawing (on ice, takes a while) and freezing doesnt do anything major to it, provided you worked clean and got not RNase contamination. You might see a neglectable amount of chemically degraded RNA.

In our experience, it is best to have a designated RNA bench, RNA pipets, RNA tips, basically a fully equipped workplace that is totally RNase free. Everything on it is wiped regularly (every few weeks) with RNaseZap, gloves are only touched by the wrist, never by the fingers. If you follow these measures from the point of extraction on, not much will happen to your RNA.

Storing with EDTA might impair future reactions.

I have put RNA in alcohol as a precipitation and stored it at -80 oC for one or more years. You can take it out and process it as a normal precipitation. I have used such RNA for RT-PCR/cDNA cloning. I know the reaction product may be less than fresh RNA but we cannot always have a perfect experiment..

It is better but more expensive to keep cDNA because is more stable.

Keep everything in -80 and think twice before you remove it from freezer, every cycle of freezing-defreezing will influence quality of RNA

RNAse free, store at -80°C and you can do this with you Agarose gel http://www.ncbi.nlm.nih.gov/pubmed/22222980

It also depend a lot from the amount and the dilution of RNA. I work with few cells at a time, and as a rule we try NOT to store. If we have to, -80, no thaw-refreeze, and for as few days as possible. Whenever possible, I don't stop at the RNA step but do the aplification/RT step or whatever comes down the line that offer a more stable product. With larger amount of material, though, mid to long term sotrage at -80 in RNAse free buffer, possibly with RNAse inhibitors, is not a problem.

As many of the above, I recommend frozen storage in water under nuclease-free conditions. The pH is then slightly acidic. If you use a buffer, you should have a pH of 5.5-6. EDTA improves the stability by removing Mg2+, but may cause problems with your downstream process. We have tested, in a methods course, to do RT and qPCR on RNA that have been sitting in water for 5 years at minus 20°, with good results.

In my experience, I found that RNA is stable even at -20°C for a long time in a simple RNAse free buffer. However it is absolutely necessary to avoid freeze-thawing, as this is really deleterious for nucleic acids, both DNA and RNA. So you may get small aliquots and store them.

In principle, if the RNA is stored in sterile buffer at pH

This may also help: http://www.biomedcentral.com/1471-2199/10/21

I recommend you have fresh RNA whenever possible . The quality of your results will vastly improve. If storing is required, I noticed that both, -20C or -80C, do the job, the key is to avoid many freeze-thaw cycles. After storing, run your samples to check the integrity of the rRNA which will reflect the integrity of the mRNA.

RNazap is a very good reagent to keep Rnases out, however it can be expensive. Alternative solutions can be used, such as NaOH 0.1M.

Use nuclease free water or DEPC-treated water, as much as you can.

Have a separate bench for RNA with its own equipment, if possible.

I agree it depends on quality of your RNA, mainly the concentration. I had worked with RNA samples stored for 6-12 months in buffer RNAse-free, but with higher concentrantion of RNA. Another experience is store RNA in ethanol (75%) at least, at -20 C, and to use, you may centrifuge and resuspend in buffer RNAse free.

RNA later from Ambion/Qiagen has worked well for colleagues of mine. It depends on what RNA you are targeting (mRNA being the most labile) and what sample type you are working with.

http://www.qiagen.com/products/rnastabilizationpurification/rnalaterrnaprotectsystems/rnalaterrnastabilization.aspx

RNA extracted and stored at -80C is working good for 2 years for us. So it all depends on how RNAse free you do the extraction and the temperature consistency of the deep freezer..

I always suggest my fellows to synthesize cDNA on the same day mRNA is extracted. At least I do not need to worry about degradation of mRNA. DNA is very resilent to degradation in cold environment and that is why scientists can sequence gDNA from ancient mammals kept in Siberia for million of years.

In our experience RNA extracts can be stored for several months at -80. Thawing and re-freezing must be absolutely avoided.

I keep my RNAs at -80ºC (except the ones I am using every second day) they are resuspended in water and some of them (actually a nice set from a tissue distribution analysis) are from 198. They are still fine, even if they went from Europe to the US and then back.

best

Jose A.,

As with many of the scientists above, I have stored RNA in water at a variety of concentrations at -80C for several years with no apparent loss of amplification. If I know I'll be using the RNA frequently, I store it in aliquots to limit freeze-thaw cycles. But I've also seen other message boards where people claim they store RNA in buffer + RNAse inhibitors at 4C for extended periods of time (up to 2 years, I believe) with no measurable degradation. So ultimately, I think RNA is more resilient than we give it credit for unless RNAses or divalent cations are present.

I am wondering what would be a nice test to check the quality of the RNA you still have in your tube?

Best

in reply to Casedei Nicolas comment the easiest answer is run a small sample of the RNA through a BioAnalyzer. You can detect if there is a fraction of degraded RNA as small as 5% (I have done it with kinetics of RNAs that were degraded during apoptosis where the amount of RNA degraded had previously been quantified del Prete, M. J., Robles, M. S., Guio, A., Martinez-A, C., Izquierdo, M., and Garcia-Sanz, J. A. (2002) Degradation of cellular mRNA is a general early apotpsis-induced event. FASEB J. 16, 2003-2005 manuscript 2010.1096/fj.2002-0392fje

and we were able to detect 5% of degradation in about 15 min.

I never heard about the "bioanalyzer". But it looks really nice tool. I think I will have a closer look on it, because our group have one!

The bioanalyser is a perfect tool also to measure the RIN algorithm (RNA Integrity Number)!

Yes, but RIN values might change if 28S and 18S RNA peaks shift (migrate faster or slower, even undergraded), causing a decrease in RIN due to misreading of the peaks by the algorithm.

In addition to the Bio-analyzer, you might also try the so-called 3'-5' assay, whereby primers are designed near both ends of the RNA. After reverse transcription with oligodT primers, you can assess the ration between the 3' and the 5' primerpairs. A ration of 1 means that the RNA strands are intact. A lower ratio indicates partial degradation.

With this assay you can directly assess whether the sample quality may bias quantification. RIN numbers give you an indirect indication, as it is based on the quality of ribosomal RNA and not on the mRNA itself.

I kept my RNAs at -80ºC under 3M NaAC (1/10 V) and ethanol during three years. After that I used it in RT-PCR.

If you are really concerned about the quality of your total RNA, you can check by running it on a denaturing gel, although in most cases this would constitute an unacceptable loss of a large fraction of your RNA sample. Here is a good description of the process (from a Clontech manual):

examine RNA integrity by electrophoresing samples on a denaturing, formaldehyde 1% agarose/EtBr gel. Analyze results according to the following criteria:

-> Intact total mammalian RNA typically exhibits two bright bands—corresponding to ribosomal 28S and 18S RNA—at ~4.5 and 1.9 kb, respectively, with a ratio of intensities of ~1.5–2.5:1.

-> Mammalian poly A+ RNA appears as a smear from 0.5–12 kb with weak ribosomal RNA bands present. The size distribution may be smaller (0.5–3 kb) for nonmammalian species.

-> If your experimental RNA appears smaller than expected (e.g., no larger than 1–2 kb), as a smear

The article to read on that would be:

Quantification of mRNA using real-time RT-PCR

Tania Nolan, Rebecca E Hands & Stephen A Bustin

Nature Protocols 1, 1559 - 1582 (2006) Published online: 9 November 2006

doi:10.1038/nprot.2006.236

The theory is to design primers to both 3' and 5' ends of your target transcript and compare the efficacy of amplification. A ratio of 1 would indicate intact RNA (that both 3'-end and 5'-end of the transcript is intact) as reflected by the cDNA involved. But, one must always weigh the priming strategy used when making cDNA beforehand, as oligo d[T]n priming will likely always show "3'-end bias", and, therefore, always give a better 3'-end amplification... (and would be an unfair representation of the intergity of the 5' end if the RT enzyme was not robust enough to travel more than 1200 or 1500 bps before 'falling off')...

I have had great success storing RNA at 4C for up to (and exceeding) 3 years in nuclease-free water containing 0.5 U/uL RNAseOUT, without signs of declining response to RT-qPCR. The more dilute the RNA from this point (after storage), the poorer it performs. E.g., standards diluted from the initial stock RNA (starting as diluted 1:10 with nuclease-free water and RNAseOUT) fare worse and worse over time the less concentrated the standards are.

Re-extracting RNA from 50% ethanol precipitates always introduces other worries. Although, it is technically a safe way to store RNA at -80C.

Use an RNAse inhibitor during storage is what I would suggest - and nuclease-free water. Freeze-thawing of purified RNA is not a big factor with RNA integrity. It is the thawing of tissues from which the RNA is extracted from that exposes the RNA to RNases, that is the major culprit of RNA degradation. The sooner the RNA is in an RNAse-free environment, the better. RNAseOUT is expensive, but it does the job -- even at 4C storage. RNA is tougher than most think it is, and with reasonable manipulation (RNase-free as possible and using an RNase inhibitor during storage in nuclease-free water), it can last for years.

If you've ever read about freeze-thawing of genomic DNA (long strands), it can withstand as much as 40 freeze-thaws without loss of banding pattern on gels, etc. Yield is reduced after freezing (~25% loss of yield) but overt signs of "degradation" per se due to freeze-thaw is largely a myth. It is indeed DNases and RNases that we need to be worried about. pH and divalent cation-mediated self-cleavage of RNA is a concern when the 2' hydroxyl is deprotonated at pH>8 or so, and cuts phosphodiester bonds - so citrate buffers and nuclease-free water containing 0.1 mM EDTA can help limit nucleophilic attack of RNA on itself as well. RNA itself will help buffer the water pH to below 7 generally (if concentrated enough) -- so these are all things to consider.

Once you find a ritual that works 100% of the time - stick with it.

I have samples collected in November 2012 and stored in 95% ethanol in -80, then later transferred to liquid nitrogen while still in 95% ethanol. From the discussion above, am lighting up to the possibility of getting enough RNA for amplification and sequencing.

Try not to store RNA, convert it into cDNA and then you can store this cDNA at -80 degrees for quite a long period of time (we have used 3 yrs old cDNA stored at -80 in my experiments). If otherwise not possible, one can store RNA in TE buffer with preservatives (glycerol/dmso/ethanol) added, if needed, at -80.

To enhance the exploitation of exteracted product: using autoclaved microtube, tube & etc, DNAase RNAase free water, RNase inhibitor, Refrigerated centrifuge and quickly coverting to cDNA or store at -80

hey ppl please give me a protocol to precipitate RNA to store it in minus 80

Himanshi

You can ppt your RNA using 1/10 volumes of 2.5 M LiCl (Lithium Chloride) + 2 volumes of absolute ethanol and keeping the mixture at -20 degrees for 30 mins. Centrifuge the mixture at 10000 rpm for 5 mins at 4 degrees. ( wash the pellet if required with 70% ethanol)

Dry the pellet and store at -80degrees

Hi! 

I had a problem in my lab I want to know your opinion. In fact, this monday morning, i realized that the -80 was open since  fiday and the temperature droped drastically at -5 °c. i want to know if the quality of RNA and DNA stored in that fridge could be affected

I agree... it could be worrisome but a test should be performed 

@Arnaud: Your DNA must be fine. However, you should check the RNA aliquots before proceeding. Good luck !