Your problem should not happen if RNAlater is used correctly. Your tissue samples must be stored at 4oC for 2hr to overnight before freezing. If you are collecting solid tissue, one dimension must be

Hi Lawrence, thank you for your reply.

Yes, we weighed the tissue and used 1: 10 (w/v) for both RNALater and Trizol. We are contemplating using the Purelink kit, but haven't done so yet.

Hi John,

how did you store the RNAlater samples (RT, 4°C, -20°C)? I only observed loss of RNA (drop in concentration), if RNAlater freezes. However, I have stopped using RNAlater and store homongenized samples directly in Trizol at -80°C instead. This way I have made very good experience (in both concentration and RIN).

Also, how did you store your samples after adding RNA later. They recommend that you hold them on ice for 24 hours instead of putting them directly into the freezer. We dont typically have issues with RNA loss with RNAlater as long as we follow their directions.

for cell culture samples we have always lyzed in Trizol and froze for future RNA isolation. however when using RNAlater, I stored the samples at 4C for better impregnation before freezing to -20C or for long term at -80C.

Hi John! It is not usually see this decrease in RNA concentration. I have used this method to transport samples between cities and I had not any problema. I prefer use Trizol method for preserve the samples that I am going to use for RNA isolation, keeping them at -80º. Some kits form Quiagen are so good too and you can freeze the cell like a pellet "in dry" at -80º.

If you can not resolve this problema, talk with the company.

Hi John, I have employed both methods and experienced no significant differences when manufacturer's instructions were followed. However, as Mario and Joseph pointed out, if you freeze the sample right after the addition of RNAlater solution you might experience decrease of RNA concentration.

Hi John,

Mario and Joseph make some interesting points. I personally wasn't aware of the "24hr hold-on-ice before freezing" practice (I held for a couple of hours for my tissues which were already partially disrupted), but I sure will try it in future.

In my experience, there is a general understanding that RNAlater can interfere with TRIzol extraction procedures. The simple tip I learned was to centrifuge (not top speed) the sample before adding TRIzol in order to remove as much RNAlater as possible from the prep (take steps to maintain an ice-cold sample temp during this process).

The 10-fold drop you describe seems quite drastic, so I would definitely take as much care as possible at the RNAlater removal stage. Other contributors may have further tips, I can see from the suggestions so far that different labs are taking different approaches.

Good luck!

Paul

Your problem should not happen if RNAlater is used correctly. Your tissue samples must be stored at 4oC for 2hr to overnight before freezing. If you are collecting solid tissue, one dimension must be

I have no issue with RNA later. RNA yield was really good from the samples I stored in RNA Later. Infact as you asked my samples were incubated at least for 24/ 48 hours at room temp and then were used to isolate RNA. The yield and RNA integrity was wonderful.

I also used RNAlater and it worked fine for me. But as already mentioned before, I stored my cell pellets at -80, just to be sure.

Another alternative to Paul's RNAlater removal method that is used in my lab is to place the tissue sample in sterile ddH2O for a minute and then tranfer it to Trizol.

For storage of cell lines for RNA extraction we prefer to freeze directly in Trizol up to 3 months.

I advice you to use phenol:chloroform:isoamyl alcohol (25:24:1) to separate RNA/DNA/proteins if you are only using chloroform. It increases the quality and amount of RNA extracted. (It is better if you buy it instead of trying making the solution)

You should not have any problem as long as you are using the proper volume of RNAlater buffer that suit your sample size. The reagent allows permeation of tissues to preserve the RNA and stop the regulation process. Depending on the subsequent technique and when you will perform your next experiment you can keep your sample in the RNAlater buffer in various temperatures.

Dear John,

I am not sure we have ever done a head to head comparison but my impression is that our yields were very similar after we had switch to RNA later from liquid nitrogen. I am pasting below a reference that might be helpful.

BMC Genomics. 2004 Nov 10;5:88.

Comparison of frozen and RNALater solid tissue storage methods for use in RNA expression microarrays.

Mutter GL1, Zahrieh D, Liu C, Neuberg D, Finkelstein D, Baker HE, Warrington JA.

Yes it is usual when you are holding for 48 hours RNAlater buffer. The intracellular RNAses act to fragment the strands. After all it is a biological system and not a mathematical system.

We performed a proper RNA stability study wherein sputum samples from MTB non-infected subjects were pooled and spiked with known CFU numbers of BCG grown in broth to reach a final concentration of 10E7 CFU/ml of sputum. The different aliquots of 1 ml of this sputum were then treated as a test specimens. We compared 4 different methods of RNA preservation - TCEP i.e. tris (2-carboxyethyl) phosphine hydrochloride, RNA protect, RNA later and Trizol and results were compared to specimens stored without any preservative. Spiked sputum specimens were stored at 3 different storage temperatures viz. in liquid nitrogen, vapor nitrogen vs. freezer (-80). Four different test sets were analyzed (in duplicate) for RNA stability at different time points like same day, at the end of 1 week, at the end of 2 weeks and at the end of one month. Results indicated that Trizol is better RNA preservative compared to TCEP, RNA protect and RNA later. Among different storage conditions tested, storage in liquid nitrogen was found to be the best followed by storage at -80 in freezer.

Thanks very much to all of you who replied to our query. I see a few doubts on protocol arose. Here's the trial protocol.

RNA Later protocol

Gill tissue dissected and processed using 3 different extractions:

1. Trizol 1: 10 w/v and processed immediately.

2. Trizol 1: 10 w/v, homogenized, held in –80 for 3 days and then processed

3. Held in RNA Later 1:10 w/v overnight in refrigerator, then either

3.1 Held at room temperature for 3 days (diced and homogenized)

3.2 Held in refrigerator for 3 days (diced and homogenized)

3.3 Held in freezer at –20 for 3 days (diced and homogenized)

3.4 Held in freezer –80 for 3 days (diced and homogenized)

All RNA Later samples centrifuged for 2 min at 7000 rpm and 4 C, excess RNA Later removed, not fully with homogenized samples.

Trizol added 1: 10 and processed strictly according to manufacturer’s protocol.

In precipitation phase for RNA Later samples we noted a colloid like drop (instead of usual transparent pellet on tube wall). This took on pellet like aspect after washing and drying in dryblock. Not possible to remove all washing ethanol with RNA Later samples without disturbing colloid like pellet.

Qubit quantification revealed 10-fold reduction in RNA compared to other treatments

The gels and their descritpions are in the attached files.

John,

Even small amounts of RNAlater can cause problems with Trizol. Question: are you recovering all of the homogenized material? If not, cut the RNAlater with PBS 1:1 or 1:2 before spinning, and then get rid of all of the RNAlater you can see. RNAlater will increase salt concentration and the pH, which can decrease the efficiency of RNA extraction but will definitely decrease the dissolution of isopropanol with the aqueous phase. You will ppt more salt to boot. If you can't get rid of all visible RNAlater in the tube, mix in a 1:1 mixture of water and isopropanol into the tube after you've added the proscribed amount of isopropanol into the extracted aqueous phase; add enough until the solution becomes clear. This should help increase the yield from the precipitation, at least. Good luck.

The correct thickness for RNAlater is 0.5 cm, not mm. 

Good catch, Chris.