It is unclear at this point whether or not the change in temperature has impacted your samples. Since RNA is much less stable than DNA, and you didn't place your samples in any reagent to preserve the integrity of your RNA, it may be fair to say that you may lose it. How did you process the tissue before storing it at -80? What kind of tissue are you working with? If I were in your position, I would process a few samples (ex. Control vs treatment) to see if you are still able to extract the RNA. If you don't get the RNA, you didn't waste too much time/reagents/etc. If you do get good RNA extraction, then the temperature change might not have changed anything. Of course, to verify your results, you would need to repeat the experiment. This would firmly validate that the temperature has no effect if the results are reproducible.

I would not say that you lost your RNA, but it might be degraded to some degree.

as above said its dependent on the samplle type the storage time period.... as for as the RNA extraction method we are using -60 for storing....

i could only say go for isolation as soon as possible... and all the best...

As above said, most probably your RNA will be degraded to some extent. Unfortunately there is only one way to check it and is to extract RNA from a few samples. I would suggest that after RNA extraction you run an RNA chip from Agilent to determine RNA integrity (https://www.chem.agilent.com/Library/usermanuals/Public/G2938-90037_RNA6000Nano_QSG.pdf). This way you would know without doubts if your RNA samples are still usable for your experiments.

All the best.

Hi folks, thanks for your responses. There at least seems to be some hope for my samples.

I'll test the RNA quality and post the results, might be helpful for others.

@Rohan My samples unfortunately are rare so there is no way around. Who would've guessed that the new freezer causes problems. From now on I'll use RNA later or similar reagents to prevent such annoying problems.

I doubt that temporary exposure of frozen tissue to -18 °C will cause problems. Indeed, extracting RNA and doing QC is the way forward. FYI, I refer to one of our papers to assess RNA quality and determine impact on gene expression results.

Article Measurable impact of RNA quality on gene expression results ...

Hi Philipp

If the tissue is in trizol, then RNA will be perfectly intact in -20 degree C. Absolutely no need of RNAlater. So don't worry. I am telling it from my personal experience.

Good luck for your experiment...

Hi Sandeep,

my tissue was not stored in trizol..

But we are going to analyze them soon, then we have certainty.

Cheers,

Philipp

Hi all, I am finally able to solve and finally close this 'thread'. We analyzed the tissue in question and were able to extract high-quality RNA (verified by Bioanalyzer runs). The extractions were used for the construction of cDNA illumina libraries and are on the machine for sequencing right now.

Thanks for your kind answers!

I hope this QA thread might be a help for others in the future.

Best,

Philipp

That has been a very interesting post. In my opinion it made not much sense to assume the RNA would be degraded. What enzyme works at -18C at a rate to allow for substantial RNA degradation? I think there is none. But does this mean samples could be stored in a -20C freezer? How close to the freezing point can one go before severe damage sets in? Since a huge number of samples are stored worldwide and lots of people seem to like the idea of RNAlater or Trizol storage, this thread becomes very important.

I take away from this discussion: if you purely collect the tissue for extraction, RNA is this case, deviations from the -80C can be well tolerated and the decisive factors influencing RNA quality are the time before it was frozen and the way the tissue is disrupted for extraction. If you want to use the tissue for more such as histology, stainings etc. temperature fluctuations may be more of an issue. But the same thing applies: the way the tissue was frozen and the way it was sectioned and processed for staining may be more relevant for the quality of the outcome than temperature fluctuations.

Thanks everyone for this insightful discussion.