Do not use RNAlater

RNAlater is a solution of concentrated Ammonium Sulphate salts with some preparations also containing Cesium Sulphates. It works by denaturing RNAase proteins. It is not a tissue fixative.Plant and animal samples placed in RNAlater solution immediately undergo two major reactions which compromises the transcriptome you are attempting to isolate

1. There is an osmotic cell response to the high external salt concentration with water moving out and cell desiccation. This gives a response of new transcripts produced related to salt stress ( it takes 10-20 minutes for a eukaryotic cell to respond to a stimulus and start transcription). These new transcripts pollute your targeted, desired transcripts.

2. The tissue in RNAlater immediately starts dying (necrosis not apoptosis) even if stored cold. With the start of cell death there is a molecular response (as in 1.) including new transcripts (in attempts to repair, destroy insults etc.) and new RNAases. Again a new transcriptome is produced.

Is RNAlater useful for any experiment-probably not.

• The best way to store RNA long term is in ethanol at -80C
• You then remove spin down and wash with 70% ethanol
• In the past I have stored RNA from biopsied tissue samples this way for > 1 year without any degradation

Hello, Agreed with above answer. Also you can use vacuum concentator (see below info) for drying your RNA samples and then you can store from -20 to -80 for a year or more without any problem.

https://www.sigmaaldrich.com/catalog/product/sigma/z368202?lang=en®ion=AM

You can freeze RNA directly at -80C for long term storage ...

But you can also make cDNA and store at -20C, in this way you can store more stable molecule.

thanks.

Try not to make the cDNA too much in advance. Better to store the RNA and make the cDNA when you require it

Laurence Stuart Dawkins-Hall Thanks for your recommendation...So i should extrect pure RNA and suspend it in 70 % ethanol and store it in -20 0C like we do for DNA preservation? what if we want to preserve the bacterial sample for later RNA extrection and transcriptomics?

Thanks to all for qiuck and valuable suggestions.

Hi there. Resuspend your RNA in 3 volumes of molecular grade ethanol and 1/10 vol 3M sodium acetate pH 4-5. So if your aqueous extracted RNA is say 50ul you would add 5ul of sodium acetate and 150ul of ethanol. Vortex and then place st -80C for long term storage

To recover after a few months spin for 15 min. Remove most of supernatant. Vortex pellet with 0.5mls of 70% ethanol at room temp (to remove salt effectively). Spin for 5 min. Take off all of the ethanol. Spin for a further 1 min to bring down residual ethanol. Remove residual ethanol with p10 tip. Air dry on ice for about 15 min. Resuspend say in 50 up aqueous solution to use

RNA should always be stored long term st -80c not -20c. Storing as a pellet is ok. Storing in Lysis buffer prior to extraction better still. But to store already purified RNA ethanol precipitate.

Laurence Stuart Dawkins-Hall thanks

For storing pellet, the best way to go is in RNAlater . If you don't have RNAlater on hand, then store it in RNA lysis buffer as Laurence Stuart Dawkins-Hall suggested. We have some samples that was lysed in RNA lysis buffer (Qiagen/Omega Bio-Tek mini kit or similar) and stored in -80C for 8+ years and still able to extracted RNA with minimium degradation.

Laurence Stuart Dawkins-Hall will there be any effect on gene expression if i suspend and store RNA in ethanol (-80 0C)? why do you suggest not to make cDNA and store it instead of pure RNA (as cDNA is more stable)?

Thanks for your valueable suggestions.

Hi Shailesh, some important considerations to store RNA must be kept first in our minds that the RNA is stable at -80C only if the divalent cations are not present and the pH must not be too high (above 8 or so) and too low (below 3 or so). A mild acidic pH is always ideal for RNA storage. TE buffers are not much safe to store RNA, they are good for DNA / cDNA storage.

So the choice of an ideal RNA storage solution must cater 2 things;

The chelation of any possible divalent cations (as cations induce heat-mediated RNA cleavage) and presence of a low pH (as in high pH solutions, RNA bases are readily hydrolysable.

In this entire scenario, if you can avail this following solution it would be much appreciable rather preparing your own solutions;

https://www.thermofisher.com/order/catalog/product/AM7001

You can even use this as your elution buffer by replacing with elution solution of your RNA extraction kit for all of your RNA extractions and later directly can store them at -80C. RNA storage in nuclease-free water alone will definitely reduces shelf life of RNA over time even at -80C.

But, also you can synthesize cDNA in bulk and can store in aliquots in the concentrated form, and then dilute as per your experimental requirements by taking out the aliquots one by one. I have never faced any problem in this as well.

Regards...

using an adequate buffer (e.g. TE) and an RNase-free system (tubes, inhibitors)

• No. Unless cDNA is column purified it is not more stable than RNA. This is a myth
• If however you column purify and not just make then yes the impurities you remove which can contribute to degradation will not be there
• That said, it is generally considered best practice to make cDNA just before you use; If at all possible for quantitative work

Thanks everyone for the helpful suggestions.

Have you considered DNA/RNA Shield? It's much less viscous that RNAlater and you do not need to remove the solution for further downstream processing. You can find information about it here:https://www.zymoresearch.com/pages/dna-rna-shield

If you have any further questions about it, let me know and perhaps we can connect.

Do not use RNAlater

RNAlater is a solution of concentrated Ammonium Sulphate salts with some preparations also containing Cesium Sulphates. It works by denaturing RNAase proteins. It is not a tissue fixative.Plant and animal samples placed in RNAlater solution immediately undergo two major reactions which compromises the transcriptome you are attempting to isolate

1. There is an osmotic cell response to the high external salt concentration with water moving out and cell desiccation. This gives a response of new transcripts produced related to salt stress ( it takes 10-20 minutes for a eukaryotic cell to respond to a stimulus and start transcription). These new transcripts pollute your targeted, desired transcripts.

2. The tissue in RNAlater immediately starts dying (necrosis not apoptosis) even if stored cold. With the start of cell death there is a molecular response (as in 1.) including new transcripts (in attempts to repair, destroy insults etc.) and new RNAases. Again a new transcriptome is produced.

Is RNAlater useful for any experiment-probably not.

If you are trying to preserve in-situ samples I'd recommend flash freezing in liquid nitrogen in the field ASAP. Can store this way or in -80 back in the lab until performing extractions.

Do not use RNAlater.