Degradation starts as soon as soon as you thaw your samples. Make sure to work sterile and fast to complete RNA isolation the soonest possible. I would prefer to separate buffy coat cells immediately after blood collection and lyse the cells in GuSCN Buffer or Trizol. The lysate can be stored at -80°C with the risk of RNA degradation being minimized, because RNAses are denatured. I agree with Kristin that you should keep your samples as short as possible at -20°C.

Hope this helps! Good luck!

I agree with Theodora and Kristin. 

I would also recommend to train the RNA extraction protocol with some "disposable" samples. Knowing how to do every step of the protocol by heart allows you to complete the RNA isolation as fast as possible. Sterile, clean and fast working is crucial for RNA isolation. 

Good luck with your RNA extraction.

Dear colleagues,

Once I had to work with freezed at -80 RNA samples, without using of RNA later solution. The samples were purified from fresh frozen colon tissue with mini spin columns based kit (Ambion) and elute with sterile deionized water (MilliQ). RNA samples were stored for 1 year (without of freezing thawing cycles). qPCR gene expression analysis showed that in some cases, the dCq values were not such as when using fresh (not frozen) RNA samples. For example, GAPDH dCq values were for 2-3 cycles higher than earlier (before freezing). At the same time storage did not affect the CYP1A2 dCq values. Be careful, some targets are more susceptible to degradation during storage of RNA at -80. I hope that my experience will be useful.

Dear Hanieh,

Regarding the blood - it will likely be impossible to get good RNA out of that now. Freezing will make the blood hemolyze (pop the erythrocytes=red blood cells, which have loads of RNA but also loads of RNases). It depends a little bit on what you are trying to look at, but I'd say that you really need to extract the PBMCs (leukocytes=white blood cells) from the blood before freezing them. In particular, if you are looking at microRNA in serum/plasma, you must separate this from fresh blood as lysed red blood cells will release massive amounts of microRNAs that will completely mask any serum/plasma microRNAs.

Re tissue - it is ok to store tissue in RNAlater for up to 1 month at 4 degrees and at -20 for long-term storage:

http://www.qiagen.com/gb/resources/resourcedetail?id=38566945-e96b-46a4-a287-ec026849da4b&lang=en

It is important though to give the RNAlater time to permeate tissue at RT or 4 degrees before transferring to the freezer (see the handbook above).

Andrey,

the key to getting good RNA out of tissue that has not been put in RNAlater is that you need to flash freeze immediately upon collection in liquid nitrogen. You then need to make sure that your tissue does not get thawed at any time before the RNA has been extracted (once you added the ethanol in column purifications or trizol in phenol extractions). For example, if you use a pestle&mortar to powder your tissue, these have to have been pre-cooled on dry ice before use. You can still transfer liquid nitrogen flash frozen tissue into RNAlater, simply add the RNAlater to the frozen tissue and leave to thaw&permeate overnight.

Kristin is wrong to suggest that sterile conditions when collecting tissue samples will prevent RNA degradation - there are huge amounts of RNases in each cell, so any potentially added RNase at this point is negligible. The cellular RNases will start working as soon as you have taken the tissue sample or the test animal is terminated (whichever is first). So if it takes you >30 min to get a particular tissue because you have to perform another procedure first or because the surgeon has to close up the patient before they can give you the tissue sample, you may end up with RNA of not so good quality...

You only need to start worrying about RNase contamination once you have extracted your RNA!

Yes, But for best results, a snap freezing in lquid nitrogen prior to -80oC storage is essential.

Thanks for your suggestions ..all of them were so helpful...fortunately,I have put tissue samples in RNA later after collection ..I am just worried about separating bufficoat from blood...I want to know whether it is possible now(after it has been frozen),since I havent seprated it immediately after collection...Dear Petra, fortunately I Dont want to work on microRNA..I think Ishould be so optemist..in this way,the quality of my RNA will increase...

Hello Petra, i going to collect tissue samples from hysterescopic examen in hospital that is too far. I haven't got RNAlater, but do you think that RNA will be ok if collect the sample into trizol and inmediately put this in dry ice? Thanks a lot!

Dear Soledad,

You could make your own RNA storage solution (RNAlater is a brand name for the commercial product) as described here in the attached link if you have these chemicals available.

Alternatively, if the sample is very small (ie from a needle biopsy), so the cells will dissolve easily then the trizol should work. Trizol = Acid guanidinium thiocyanate-phenol-chloroform extraction, so does denature proteins including RNases and later centrifugation separates the protein phase from the DNA and RNA later.I doubt that you would need the dry ice in this.

However, if your tissue is larger pieces and particularily if it contains lot's of extracellular matrix, I would think that the trizol would not be efficient enough in disrupting and permeating all the cells quickly. You would perhaps need to physically make the tissue into a paste by squeezing through a mesh or something.

However, I have no actual experience in doing this, so what I have written is based entirely on my knowledge and could be wrong!

In your shoes, I would try and do a test-run with similar tissue from either mouse/rat treated in the way you plan to use to see if you can get comparable RNA to fresh tissue.

https://www.protocols.io/view/RNAlater-Recipe-c56y9d

For DNA methylation study, which is the best way to collect and store tumor tissue for future work . 1. Formalin fixed 2. Liquid nitrogen 3. RNA later??????

Hi Lokesh,

I'd vote for #2. You can get DNA out of formalin fixed tissue, but it's not optimal. Long term storage at -80 works okay for preserving methylation according to these guys: Article Blood DNA Yield but Not Integrity or Methylation Is Impacted...

I choose also #2. The DNA obtained from Formalin fixed tissues are mostly degraded. Therefore in Liquid Nitrogen frozen tissues will be better.

How long can I store my tissue at -80 C before RNA extraction without affecting the stability of RNA ? At maximum one month or two

We have gotten excellent RNA integrity numbers after several years storage at -80