Hi Gareth,

you have some very relevant questions. Caution is off cause at the core of generating good data, especially when it comes to sequencing.

So lets see:

Storing RNA as an EtOH precipitate is the perfect solution for long term storage, or for shipping samples to ie. a sequencing provider.

However for short term storage (several months) keeping the RNA in water, or even better a low TE buffer with a bit of RNAseOut added, aliquotted and stored at below -70 Celcius is quite adequate.

Glycogen: when precipitating low conc nucleotide samples (RNA, DNA) you need to "trick" the solution into reaching the critical precipitation conc point. By adding nuclease-free glycogen to your precipitation reaction the critical mass of sugar-backbone is driven up, allowing your scarce sample to precipitate out.

Glycogen does not interfere with the downstream reactions you mention.

Salt from precipitation: make sure to wash your precipitate twice in EtOH(70-75%), before drying (be very carefull not to overdry!, usually 5 min room temp is enough). The remaining salt (NaOAc) is not a problem downstream.

Wishing you good luck and excellent data!

Anne

Hi Gareth,

you have some very relevant questions. Caution is off cause at the core of generating good data, especially when it comes to sequencing.

So lets see:

Storing RNA as an EtOH precipitate is the perfect solution for long term storage, or for shipping samples to ie. a sequencing provider.

However for short term storage (several months) keeping the RNA in water, or even better a low TE buffer with a bit of RNAseOut added, aliquotted and stored at below -70 Celcius is quite adequate.

Glycogen: when precipitating low conc nucleotide samples (RNA, DNA) you need to "trick" the solution into reaching the critical precipitation conc point. By adding nuclease-free glycogen to your precipitation reaction the critical mass of sugar-backbone is driven up, allowing your scarce sample to precipitate out.

Glycogen does not interfere with the downstream reactions you mention.

Salt from precipitation: make sure to wash your precipitate twice in EtOH(70-75%), before drying (be very carefull not to overdry!, usually 5 min room temp is enough). The remaining salt (NaOAc) is not a problem downstream.

Wishing you good luck and excellent data!

Anne

I used to store DNA as ethanol precipitation as you described in a single tube and than take an aliquot corresponding to the amount I wanted simply after vortexing the precipitate. So you centrifuge only the volume corresponding to the amount of RNA that you are going to use in that moment keeping the stock apart. After centrifugation it is better to wash the pellet with 70% ethanol for few seconds. I used glycogen to precipitate low amount and never had problems for the further steps. You add before the ethanol in your samples The high molecular weight of glycogen helps the precipitation of the nucleic acids..

I would recommend you to store the purified RNA in water at -80 deg C. If you would like to obtain a high quality RNA, the best method is the Qiagen kit. It also depends on the source of isolation.

http://www.qiagen.com/products/catalog/sample-technologies/rna-sample-technologies/total-rna/rneasy-mini-kit#productdetails

Hi Gareth,

Invitrogen/Life Technologies provides some tips re Working with RNA that might somewhat answer your questions: - http://www.lifetechnologies.com/au/en/home/references/ambion-tech-support/nuclease-enzymes/general-articles/working-with-rna.html

http://www.lifetechnologies.com/au/en/home/references/ambion-tech-support/nuclease-enzymes/general-articles/ten-sources-of-rnase-contamination.html

and here is some useful information re Glycogen/GlycoBlue: http://www.lifetechnologies.com/order/catalog/product/AM9515?ICID=search-product

Hope that helps :)

Cheers,

Cuong

RNA is perfectly stable while frozen. However, stability when thawed is influenced by the buffer it's in - neutral pH with a very small amount of chelator will protect RNA from trace divalent mediated cleavage in water. So, T10E1 or 0.1XTE is preferable to water.

Also, the reason the alcohol precipitate is viewed as a good idea is that any trace nuclease or 'bug' will be unlikely to affect the RNA when you thaw the sample and remove an aliquot. But in general, if you made clean RNA, storage in aqueous format at-80C is fine. Why not -20? Well, many MANY -20 freezers go through defrost cycles which is truly not what you want.

Eric (RNAlater)

I would never use TE buffer for storage since TE tends to depurinify the RNA so the RNA molecule is "cut" at positions encoding A or G bases. Esp. under long term storage I would strongly suggest storage in H2O or as EtOH precipitate at -80°C.

You may try to store the RNA at -20 in water/ethanol (2.5 vol), without salt. RNA will not precipitate. Before use, just take an aliquot you need, add NaAc up to 0.3 M, incubate, spin down, wash and resuspend in buffer. If RNA concentration is very low, carrier (glycogen or linear polyacrylamide) can be added at the same time as NaAc.

I have always stored purified RNA in RNAse/DNAse free water. No problems here and it works fine for any downstream processing.

for RNA, be careful as it could precipitate immediately but it's not sure. I would aliquot after it has precipitated. When you want to use samples simply spin down to pellet the RNA.

be careful with glycogen it could be a bad thing if you made PCR amplification or qPCR. when you resuspend the pellet, you could take 10ul instead of 30ul. just be sure to note the orientation of your eppendorf in the centrifuge because you wont see the pellet after centrifugation.

RNA in water will be degraded if you heat it. Never heard that issue about TE, but of course I'm locked away in chains here :-)

Hi Gareth

I perform extractions with a comercial kit and elute from columns with water RNase-free, so I storage RNAs at -80C, because at -20 RNases could still have some activity. You can also store them in ethanol and 0.3M sodium acetate at -20C, since I send my RNAs with cold-packs to sequencing services and they arrive OK to destiny.

I am not sure if ethanol precipitacion eliminates salts, I think you should purify it in the previous steps.

To Eric Lader:

In my lab RNA is always stored in water and when we set up experiments RNA is heated. We have never experienced RNA degradation.

Dear Eric this is something which is really hard to publish but from the experiments we did it is pretty clear. There was always an effect seen that freezing and thawing seems to degrade RNA samples quite a bit but to my experiences this effect almost vanish when we avoided TE.

Dear whitely

Answer No I

Did you use beta mercaptoethanol during the isolation procedure ?Then your RNA is not going anywhere. you may precipitate it and aliquot it at this stage can store at -20 refrigerate.

Yes your choice is right for acetate. whenever you want to use your RNA you can simply centrifuge to pellet down your RNA and then wash it with 70% ethanol, to remove excess salt, spin it down and air dry and dissolve it in diethylpyrocarbonate( DEPC) treated water or buffer.

In my experience total RNAs from human or animal viruses, stored at -80°C as acetate/etanol/water precipitate, are stable for more than 30 years. After thawing samples, just washing twice the pellet in a refrigerated microcentrifuge removes salts completely. It is mandatory to dry completely the ethanol above the pellet before resuspending RNA in RNAse-free water. Usually TE helps in solubilizing total RNA from cellular extract but for viral RNA we usually use just water. If you need to aliquot the sample, you should do it before adding acetate/ethanol because precipitated RNA is not homogeneous. We use "Yeast RNA" either to visibilize pellet in dilute RNA solutions or to stabilize scalar dilutions of RNA in quantitative RealTime PCR. For practical purposes we need to store samples "ready for use" and acetate/ethanol is not so practical and it is better to use it only for special samples as standard or precious samples. The routine way is to aliquot the RNA solutions and to store them at -80. Using quantitative RealTime PCR we have experienced no degradation of RNA for this purpuse. Obviously maybe different if RNA should be used for cloning experiment.

Thank you everybody for all the answers. I also asked Ambion (via [email protected]), and got the following very helpful answer. I hope it helps anyone else interested in this area/issue:

Thank you for contacting Life Technologies Technical Support (The Life Sciences Solutions division of Thermo Fisher). You are making your ethanol precipitate/slurry correctly by adding 3 ul of 3M sodium acetate and 90 ul of 100% ethanol to your original 30 ul RNA in water. The RNA will start to precipitate out at this point. You might be okay to aliquot the mixture if you do it immediately but my guess is that the RNA will start precipitating out as soon as you add salt and ethanol so you may want to avoid aliquoting.

When you are ready to use the RNA you will spin it down to generate a RNA pellet. Please remove the supernatant and you may wash the pellet several times in 70% ethanol (made with nuclease-free water). After each wash spin down and remove the supernatant. After removing the final wash you may resuspend the RNA in your buffer of choice. The washes with 70% ethanol will help remove the residual salts.

You may also make a salt/ethanol slurry with samples that have a low RNA concentration. You may use a carrier to help precipitate the RNA. You would add it at the same time as the salt and ethanol although I would add it before the ethanol is added. The carrier does aid in the visualization of the RNA and helps increase the nucleic acid concentration and make it less soluble to help with the precipitation. It does not physically interact with the RNA. This will not affect the resuspension protocol but the carrier RNA will be mixed with your experimental RNA. There is no way to remove the carrier.

Also, Michele Muscillo, you said you store your RNA acetate/ethanol precipitates at -80C. Most people suggest -20C. Have you found that -80C is even better?

The presence of ethanol and salts prevent the freezing of acquous solutions. You can see by yourself whay happends in either -20 or -80 with such solutions. At -80 tubes with denaturatd RNA in alcool are actually very viscous and "seems" solid. Anyway, it is a common practice to precipitate nucleic acids at -20 with acetate/etanol, therefore denaturated RNA is supposed to be stable also at this temperature. I have never compared samples stored at these different temperatures. We have experienced that scalar dilutions in water of syntetic RNA, used for calibration curves in quantitative Realtime RtPCR, give better results if stored at -80. I have viral RNA standards prepared more than 20 years ago and stored at -80 and they still work in RtPCR. That's all.

20 years and still ok - great to hear!

Thanks.