RNA later. We get optimal results for storage of small biopsies for RNA and DNa extraction for microarray analysis. NGS has similar requirements of RNA/DNA quality. In RNA later nucleic acids are stabel for several days at room temperature and extended times at -20°C. RNA later should not be stored at -80°C.

Have you considered using a mixture of melting ice and Calcium chloride? At its eutectic point, this mixture can reach -55 C. Common salt and melting ice will go as low as -21. Do not use dehydrated CACl2, since this will produce heat upon dissolution.

thanks for the suggestion, but this really needs to be something that can easily be introduced into multiple centres - so needs to be simple and manageable by non scientific staff. I was thinking more of one of the commercially available solutions for preserving RNA and/or DNA and wondered whether anybody had experience with any of them specifically with respect to getting good enough quality DNA for whole exoome or genome sequence?

Dear Jennifer,

The use of these salts with melting ice is as low tech and low risk as you can get? The commercially available solutions require lab skills, and above all, are not without risk when applied/used by uninformed, not -skilled persons. (Which I read from your reaction). Exoome is a term not familiar to me, please explain.

Hi Harrie, Sorry, that was a typo..meant to be whole exome sequencing (selective sequencing of the transcribed regions of the genome as a more affordable alternative to sequencing the whole genome).

The commercial storage solutions I was thinking of don't require any lab skills, and can stabilise DNA and or RNA (e.g. RNA later, DNAstable plus), and allow storage at room temperature until DNA extraction, but there are quite a few of them out there so I was wondering if anybody had a specific recommendation when dealing with small amounts of human tissue (biopsies) when the extracted DNA need to be of very quality suitable for high throughput sequencing

Dear Jennifer,

I think you should read my question, since it is entirely related. I am also looking for protocols for mRNA extraction, and high throughput sequencing of biopts. My questions is related to a paraffin embedded biopt of a primary HCC tumour.

Hi Jennifer,

I would definitely not recommend RT for storing your samples prior DNA extraction. Preservation of samples is better at lower temperatures since most endogenous nucleases are relatively active at RT and consequently their activities would diminish at lower temperatures. So, if you do not have dry ice, N2(l) nor a -20 deg freezer, then I'd suggest to put the biopsy samples in sterile media (RPMI, DMEM or else) without serum in the fridge. This would normally keep the cells in the tissue quite happy until your process them the next day or so (do not leave it for too long!). In my previous job, sometimes, depending when there was a lung cancer donor or surgery procedure, I would get samples at midnight, so instead of preparing the DNA or RNA then, I would put it in fresh, sterile RPMI 1640 overnight or more if necessary (up to 36 hs I've done it) at +4 degrees (fridge). I had no problems with those samples. This is low tech, can be tested in your pipeline, and if it works then you are up and running. This step was in our pipeline as a Plan B.

Alternatively, you can start the extraction (when possible) by adding the lisys buffer which normally comes or you need to add proteinase K and nucleases inhibitors. Then, in this buffer the DNA will keep stable for a couple of days at +4 deg or even better at -20 deg.

Good luck!

Hi Jennifer,

I agree with Luciano's suggestion. Of course the colder the better, but since you do not have access to liquid N2 or dry ice, I suggest finding a way to process the sample as much as possible at the time of collection. You have to find a way to bock the nucleases. So proteinase K + lysis buffer will help, but without homogenization this will only help a little bit.

You could also try including 50 mM EDTA into the collection solution because nucleases are divalent cation-dependent. This coupled with a higher salt concentration (say 1 M NaCl) will create a hypertonic solution that might dehydrate your tumor samples, which could help prevent nuclease activity. I haven't tried this, but it might be worth a shot.

Another thing worth trying would be to adapt Qiagen's "RNA later" kit for genomic DNA preps. I have never used this product, but people in my lab do and it is great for preserving RNA stability for up to 7 days at room temp. I don't know about DNA though.

Hope these help.

RNA later. We get optimal results for storage of small biopsies for RNA and DNa extraction for microarray analysis. NGS has similar requirements of RNA/DNA quality. In RNA later nucleic acids are stabel for several days at room temperature and extended times at -20°C. RNA later should not be stored at -80°C.

I also would recommend using RNAlater as samples in RNAlater can be be stored for 7 days at RT, 4 weeks at 2-8ºC, or long term at

We store biopsies (FNAB and bronchial biopsies) in RNA later both to extract RNA or DNA later on. In case of FNAB lavage the material was stored in RNA later buffer in -80°C for months, but we avoided thawing and freezing. In case of material from BAL or bronchial biopsy we store the material up to one week in 2-8ºC (in normal freezer).

Why can't the RNAlater be stored at -80C ? I haven't found this to be a problem.

Although RNAlater samples can be stored at -20C, I don't think there is any problem with lower temps. I recently extracted DNA from some mouse liver samples that had been stored in RNA later at -80C. They were fine. The important issue may be that you want to get the samples well soaked in RNAlater at a warmer (4 or above) temp since the RNAlater will not be able to penetrate into tissue that is frozen solid. You can find recipes for "homemade" RNA-later by Googling "homemade RNA-later", but I don't know if they all work completely the same.

Already Ulrich has answered our way of dealing with your situation. But if you want to store the tissue for longer time, you need to have a -80C freezer. Usually samples are highly stable in RNA later for as long as a week. Usually we collect Tissue at RNA later ( in multiple aliquot). Leave it at 4 degrees for 24 hrs. Then the long store aliquots are kept at -80 ( step wise chilling, -10 -> -20 -> -70 -> then -80) and the one that would be isolated immediately ( may within next 2-3days) are kept in -20 degrees. Any way, its better to avoid freeze thaw. Have to plan accordingly.

Hope this would help.

Thank you.

Hi Jennifer,

I am confronted to the same problem as you at the moment. I would kindly like to ask you which method did you finally use to collect the biopsy samples at RT?.

And as a general question to you all. I am also coordinating a multi center study but in our case we have access to dry ice. In your experience, what would be the best approach to store biopsy samples in dry ice for NGS and IHC purposes? Can they be stored without RNA later and are they stable enough? For how long?.

Thanks a lot!

Sandra

Yes, RNA later is the best for extraction of RNA and or DNA. Storing at -80 for long term is better but avoid repeated freeze /thaw.

My question is what is the best storage method is for protein extraction?

Thank you