It should certainly depend on the pH of your solution.

Did you chek it ?

For information the protocol preparation is :

The description in this example provides one manner in which RNAlater™ can be prepared. First, one should prepare or obtain the following stock solutions and reagents: 0.5 M EDTA disodium, dihydrate (18.61 g/100 ml, pH to 8.0 with NaOH while stirring); 1 M Sodium Citrate trisodium salt, dihydrate (29.4 g/100 ml, stir to dissolve); Ammonium Sulfate, powdered; Sterile water.

In a beaker, combine 40 ml 0.5 M EDTA, 25 ml 1 M 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 pH5.2 using 1 M H2SO4. Transfer to a screw top bottle and store either at room temperature or refrigerated.

Final concentrations:

25 mM Sodium Citrate

10 mM EDTA

70 g ammonium sulfate/100 ml solution

pH 5.2

Hi Eric,

Thanks for your quick answer. Certainly, we did not check the pH because the Ammonium sulfate was not completely dissolved, after let it cool down up to 2 fingers of cristals in tall appered in the beaker and thus, we thought that it could change the pH.

I have compared your protocol with ours, and it is just the same. Our further question is the following one: how long did you stir the solution on a hot plate? and at what temperature?. Do you check the pH even if the cristals are on the beaker?

With commercial ones sometimes also appeared mentioned cristals, and they tell you to warm up a little bit the solution. Do you usually do it before include your samples?

Thanks a lot,

Sincerely,

Oihane

Thanks Caroline, your comments are very useful!!

Hi Sokratis,

No I don't filter the solution, because I guess, you will loose salts, and also colapse the filter. On the other hand, I have never tried autoclaving it.

I always autoclave the ultrapure water (No RNAse free, because is too expensive) and use it as buffer. I do not see the problem of using DPEC treated water, and you can also filter it if you want.

Hoping this would help you,

Sincerely,

Oihane

We have been using home-made RNA later for quite some time now, it works well for Peltigera lichens, Arctic char and salmon fish. We use milliQ water to make it, filter-sterilize the final solution and pour into fresh 50 ml tubes. So far, we have reused the sterile filter twice, no clogging (sterile Nalgen filter, diameter around 8 cm).

By the way, if you intend to store your samples just briefly (a few days) in a cool place, and are going to do Trizol RNA extractions, you don't need RNAlater. You can store directly in Trizol. I would not store in Trizol for longer than 3-4 days though.

I would not autoclave the RNAlater solution as that does not efficiently inactivate RNAses and it increases the risk of contaminating the liquid.

I have heard from colleagues that DPEC treatment is not particularly efficient at inactivating RNAses. Perhaps it is better to use another form of ultra-pure water; millipore filtering system should do the trick but the system is very expensive. I think you can buy ultrapure milliQ water from that company directly, but other companies must provide similar products (hopefully cheaper).

Good luck!

See also in this regard, the recent paper in Nature Methods where they report a RNA preserving buffer: 10.1038/nmeth.2910

Reply to Sokratis:

You can autoclave about anything, but RNAses are not inactivated by autoclaving; DNAses are inactivated. People from our lab think that there is little point in using autoclaved liquids for RNA work (but at least you will have gotten rid of biological sources of RNAses).

We bake our glass and metal equipments at 200C for several hours before using it for RNA preps.

Does anyone know what the tRNA in that recipe is good for? Mysterious!

tRNA used in the hybridization buffer works as a carrier and filler molecule to take up small spaces and thereby increases the chances for a better molecular interaction between the target and the probe.

I have a question about how you adjust the pH of the stock solutions and the final RNAlater solution without possibly contaminating the buffers. Are you using the pH probe as usual? I have heat-treated my glassware at 180C for 4 h and will be making up all solutions with DNAse and RNase- free water (Invitrogen), but am curious about how I will adjust the pH whilst maintaining strict sterility.

Hi Natalie, that's a good question, one that I was wondering about recently, because I don't know that the electrode solution, the electrode storage solution, the calibration buffers etc are RNAse free.  I don't have a big wash bottle of RNAse free water.  And the pH meter lives out on the bench.

One way you might handle this is to make up a little bit bigger batch of buffer than you need, say 110% of the recipe, and "sacrifice" 10% of it (pour into a different container), keep track of exactly how much NaOH (or whatever) it takes to hit the pH target, and then scale that up to adjust the pH of the rest of the buffer blindly, i.e. without the pH meter.  You can check the pH afterward, if you pour out a little more of the "adjusted" buffer into another container.  But in this way, the pH meter, with it's questionable cleanliness, never gets immersed into the main bottle of buffer.  (and filter-sterilize when you're done).

Hi,

For the final concentrations of homemade RNA later, 40 ml of 0.5 M EDTA makes a final concentration of 20 mM and not 10 mM. However, the recipe as given will generate a higher volume than 1 L, there is factor about 1.38 (so final volume is about 1.38 L after dissolution of the salt).

What is reagent can replace ammonium sulfate in RNAlater solution?

Hi Sigit,

According to the RNA later patent, ammonium sulfate is pretty specific and cannot really be replaced to effectively preserve RNA. They tested ammonium carbonate, amonium chloride, potassium sulfate, and magnesium sulfate, these were not conclusive. They also tested cesium sulfate and cesium chloride, that only partially preserved RNA. So as far as I know, the ammonium sulfate cannot be substituted.

Best,

Audrey