Personal Note From Patrick, The Editor

Hi Reader, what temperature are your freezers set to?

Most often, there is concern about setting freezers to -70°C.

One argument is power outages - however, this only gives you 2–4 hours until a -80°C freezer reaches -70°C.

The other major concern is sample degradation... Let’s discover what the literature says:

Today's Lesson: Is Storage at -70°C Safe?

Reviewing the literature on low temperature storage

Number Of The Day

If you set your freezer from -80 to -70°C, you save between 22–30% of energy. Moreover, due to lower compressor workload, you reduce the probability of freezer failure. Currently, the most efficient models use between 5.4–8 kWh per day, depending on the model and manufacturer. When it comes to -20°C, data are much more scarce, but 1–2 kWh per day seems to be the norm. Therefore, how far “down” can we go?

Can We Safely Switch To -70°C

It was only in 1968 that a company named ScienTemp started producing freezers for the laboratory.

The story goes that storing at -70°C was the norm just a few decades ago. In 1997, the -86°C ULT freezer series we know today was introduced by Sanyo.

On the left, you see the first -86°C ULT freezer from Sanyo. On the right, a model that came out as early as 1987, already achieving -135°C. The issue is that the lower the temperature we use, the more specialised and expensive the equipment we need. This also goes hand in hand with higher energy bills and increased maintenance. This is why choosing the highest possible temperature is often the best choice — not only to save resources but also to worry less about potential incidents.

It seems that advances in freezer technology and new marketing initiatives led to -80°C becoming the standard.

However, -80°C as a set point is contingent – there is no scientific basis for it.

Assessing The Data

A missing basis for -80°C is also reflected in what we discussed in our previous lesson: several papers indicate that storing DNA at -20°C is often completely fine, especially if storage doesn’t surpass multiple years^1,2.

A really interesting paper as it investigates the addition of foreign DNA/RNA to increase stability. However, something appears to have gone wrong with the -20°C samples in the first panel. Depicted is the “long-term stability of three pDNAl formulations (TE0.1 buffer alone or TE0.1 buffer containing either carrier DNA or RNA) at 4°C, -20°C and -80°C. The values at each time point, except zero, are the average concentration values with their standard deviation for three vials each analyzed in triplicate (n=9).” by Baoutina et al.

Especially if we lyophilize DNA, this type of storage seems to be sufficient³.

However, other treatments like with Trehalose or commercial systems also allow for higher temperature storage^4,5,6.

Shown are the DNA extraction yields measured by UV spectrophotometry by Bulla et al. If you take a look at the paper you will see that their untreated EDTA blood storage looks much worse and shows some inconsistencies like higher DNA yields when storing at +4°C instead of -20°C.

We also find literature indicating that high temperature short-term storage of RNA can be successful, but degradation becomes noticeable after a few days to months^7,8.

This is why lower temperatures were preferred. However, especially if RNA is lyophilized, storage can be conducted even at 4°C and with freeze drying at -20°C in these cases^9,10.

Looking At Other Sample Types

For proteins, a set of (older) papers showed that storage at -70°C (and in some cases even -20°C) was successful^11,12.

Woodhams et al. assessed the stability of a wide range of plasma proteins with a coagulation analyser. The legend reads: "Plus = 1.5mL Micrewtube at -74°C | Square = 1.5mL Micrewtube at -24°C | Triangle = 5mL Polystyrene frozen at -74°C and stored at -24°C | X = 5mL Polystyrene frozen and stored and -74°C | Circle = 5mL Polystyrene frozen and stored at -24°C."

In some cases, like for Prostate specific antigen storage at -20°C led to a 0.9% loss, while storage at -70°C led to a 0.4% loss for each month in storage¹³.

Beekhof et al. showed that Paraoxonase-1 enzyme activity remained unchanged after one year of storage at -70°¹⁴.

Similar results were found for bacteria (S. aureus)¹⁵.

A Direct Comparison

Maybe most interestingly, there is a handful of papers that compare storage at -70°C versus -80°C directly:

Landor et al. found no significant degradation in DNA and RNA stored at -70°C, with only minor 260/230 ratio variations, all well below standard deviation ranges¹⁶.

Shown are some RNA concentrations measured with a Qubit 2.0 (ThermoFisher) by Landor et al.

Espinel-Ingroff et al. successfully recovered 6,000+ yeast and 300+ mold samples after 10 years of storage at both -70°C and -80°C¹⁷.
Bhattacharya & Nissen showed that also for plasma proteins there is no difference after 3 months of storage¹⁸.

Moreover, a non-peer-reviewed study conducted by a team from the University of Virginia found no meaningful differences in RNA quality, sequencing performance, or tissue analysis after one year of storage at -70°C compared to -80°C.

Of note, even manufacturers acknowledge this - QIAGEN officially recommends storing RNA at -70°C.

And Still...

However, there is nuance here. Cooled storage only buys us time. It does not avoid the unavoidable.

A handful of studies show that even at -80°C, certain samples can still degrade during long-term (several years of) storage^19,20,21,22.

Click to enlarge. Wang et al. depicted "Yield of DNA extraction from preserved samples across 11 groups (50 cases per year). (b) A 260/A 280 ratio of DNA extracted from 11 groups of preserved samples (50 cases per year). (e) A 260/A 280 ratio of RNA extracted from 11 groups of preserved samples (50 cases per year). (f) The RIN (RNA integrity number) values for 11 groups (50 cases per year) of RNA extracted from stored samples using an Agilent 2100. Grade A: RIN ≥ 6 and 28S/18S ratio ≥ 0.7, total yield ≥0.2 μg; Grade B: RIN = 5.0–6.0, or RIN ≥ 6 but 28S/18S ratio < 0.7, total yield ≥0.1 μg; Grade C: RIN < 5.0, total yield <0.1 μg."

Unsurprisingly, this effect is more pronounced for RNA than for DNA.

For proteins, the picture becomes more complex.

Predicting their decay is more challenging and we have to consider how long they retain ligand-binding capacity.

Several studies have shown that this varies greatly between proteins, and some even lose ligand-binding capacity below -90°C^23,24.

This shows the “average mean-square hydrogen displacement obtained from analysis of Iₑₗ(q), assuming an asymmetric two-state model” as described by Doster et al. I removed the rest of the figure panel because I doubt that anyone outside the field could meaningfully interpret it. In essence, the authors used neutron scattering (“shooting” neutrons at a sample) to directly observe how atoms inside the protein myoglobin move as temperature increases. Their key discovery is a sharp dynamical transition around 180 K, where myoglobin shifts from behaving like a rigid, harmonic solid to a more flexible, biologically active structure.

For tissues and cells, it does not get any simpler.

The literature indicates broad differences. Some tissues, especially when treated properly, can be stored at -20°C for several months, but for long-term storage, -196°C (liquid nitrogen) is often preferable.

Another influential factor we have to consider is the specific protocols applied, such as snap-freezing or the use of preservation agents. Especially for tissues or plasma, this is essential but needs to be assessed on an individual basis^25,26.

Overall, Hubel et al. put together an amazing overview of storage conditions²³. Also Kypraiou & Varzakas have reviewed diverse studies on different storage conditions²⁷.

Applying The Knowledge

In general, we can summarize that storage at -70°C seems as safe as -80°C.

Just a few decades ago, most studies compared storage at room temperature or -20°C to -70°C, concluding that -70°C was the preferred temperature for DNA, RNA, bacteria, and even tissues.

Yagüe et al. studied the storage of Staphylococcus aureus: “The median (point) and 95% credible interval (error lines) of the decay rates (slope) for each temperature and isolate combination are shown in colour while the median and 95% credible interval of decay rate averaged across isolates for each temperature is shown in grey, as estimated by the random effects in the statistical model.

Those papers that assessed both conditions also support this notion.

To convince your colleagues please also use the list of samples stored at -70°C set up by I2SL.

Still, some of the variation (and even incongruencies such as concentration increases over storage time) between studies often stems from differences in sample quality and processing, meaning there is inherent biological and methodological variation when assessing degradation.

This is Woodhams et al. once again with a strong increase of protein stability in some storage conditions – essentially an improvement in quality after storage.

That means when extremely high sample quality is required, freezing as low as possible is preferable - meaning liquid nitrogen rather than a standard freezer.

However, all freezing is associated with quality loss and even thawing can lead to apoptosis or degradation of analytes.

Optimizing storage preparation — through chemicals, snap-freezing in liquid nitrogen, or lyophilization (especially for some RNA samples) — can significantly improve stability and sometimes even allow for storage at room temperature^28,29,30.

Upcoming Lesson:

Savings in Workflows

How We Feel Today

References

1. Baoutina, A., et al., 2019. Storage stability of solutions of DNA standards. Analytical Chemistry, 91(19), pp.12268–12274. doi:10.1021/acs.analchem.9b02334.

2. Zhao, Y., et al., 2017. Effects of preanalytical frozen storage time and temperature on screening coagulation tests and factors VIII and IX activity. Scientific Reports, 7(1), 12179. doi:10.1038/s41598-017-11777-x.

3. Podivinsky, E., et al., 2009. Effect of storage regime on the stability of DNA used as a calibration standard for real-time polymerase chain reaction. Analytical Biochemistry, 394(1), pp.132–134. doi:10.1016/j.ab.2009.06.024.

4. Ivanova, N.V., et al., 2013. Protocols for dry DNA storage and shipment at room temperature. Molecular Ecology Resources, 13(5), pp.890–898. doi:10.1111/1755-0998.12134.

5. Röder, B., et al., 2010. Impact of long-term storage on stability of standard DNA for nucleic acid-based methods. Journal of Clinical Microbiology, 48, pp.426–432. doi:10.1128/JCM.01230-10.

6. Bulla, A., et al., 2016. Blood DNA yield but not integrity or methylation is impacted after long-term storage. Biopreservation and Biobanking, 14(1), pp.29–38. doi:10.1089/bio.2015.0045.

7. White, M.P.J., et al., 2024. Integrity of RNA in long-term-stored cervical liquid-based cytology samples: implications for biomarker research. BioTechniques, 76(6), pp.245–253. doi:10.2144/btn-2023-0112.

8. Jones, K.L., et al., 2007. Long-term storage of DNA-free RNA for use in vaccine studies. BioTechniques, 43(5), pp.675–681. doi:10.2144/000112593.

9. Molnar, A., et al., 2021. Lyophilization and homogenization of biological samples improves reproducibility and reduces standard deviation in molecular biology techniques. Amino Acids, 53(6), pp.917–928. doi:10.1007/s00726-021-02994-w.

10. Riesgo, A., et al., 2012. Optimization of preservation and storage time of sponge tissues to obtain quality mRNA for next-generation sequencing. Molecular Ecology Resources, 12(2), pp.312–322. doi:10.1111/j.1755-0998.2011.03097.x.

11. Woodhams, B., et al., 2001. Stability of coagulation proteins in frozen plasma. Blood Coagulation & Fibrinolysis, 12(4), pp.229–236. doi:10.1097/00001721-200106000-00002.

12. Kitchen, S., et al., 2021. International Council for Standardization in Haematology (ICSH) recommendations for processing of blood samples for coagulation testing. International Journal of Laboratory Hematology, 43(6), pp.1272–1283. doi:10.1111/ijlh.13702.

13. Woodrum, D., et al., 1996. Stability of free prostate-specific antigen in serum samples under a variety of sample collection and sample storage conditions. Urology, 48(6 Suppl), pp.33–39. doi:10.1016/S0090-4295(96)00607-3.

14. Beekhof, P.K., et al., 2012. Long term stability of paraoxonase-1 and high-density lipoprotein in human serum. Lipids in Health and Disease, 11, p.53. doi:10.1186/1476-511X-11-53.

15. Panisello Yagüe, D., et al., 2021. Survival of Staphylococcus aureus on sampling swabs stored at different temperatures. Journal of Applied Microbiology, 131(3), pp.1030–1038. doi:10.1111/jam.15023.

16. Landor, L.A.I., et al., 2024. DNA, RNA, and prokaryote community sample stability at different ultra-low temperature storage conditions. Environmental Sustainability, 7, pp.77–83. doi:10.1007/s42398-023-00297-2.

17. Espinel-Ingroff, A., et al., 2004. Long-term preservation of fungal isolates in commercially prepared cryogenic microbank vials. Journal of Clinical Microbiology, 42(3), pp.1257–1259. doi:10.1128/JCM.42.3.1257-1259.2004.

18. Bhattacharya, S., et al., 2024. Reduce energy consumption in your laboratory – switch ultra-low temperature freezers from –80 °C to –70 °C: a pilot study on short term storage of plasma samples for coagulation testing. Scandinavian Journal of Clinical and Laboratory Investigation, 84(6), pp.421–424. doi:10.1080/00365513.2024.2394981.

19. Tang, R., et al., 2022. Quality control of DNA extracted from all-cell pellets after cryopreservation for more than 10 years. Biopreservation and Biobanking, 20(3), pp.211–216. doi:10.1089/bio.2021.0052.

20. Yuwono, N.L., et al., 2022. Circulating cell-free DNA undergoes significant decline in yield after prolonged storage time in both plasma and purified form. Clinical Chemistry and Laboratory Medicine, 60(8), pp.1287–1298. doi:10.1515/cclm-2021-1152.

21. Wang, Z., et al., 2024. Assessing the impact of long-term storage on the quality and integrity of biological specimens in a reproductive biobank. Bioengineering & Translational Medicine, 9(6), e10692. doi:10.1002/btm2.10692.

22. Stephenson, N.L., et al., 2020. Quality assessment of RNA in long-term storage: The All Our Families biorepository. PLoS One, 15(12), e0242404. doi:10.1371/journal.pone.0242404.

23. Hubel, A., et al., 2014. Storage of human biospecimens: selection of the optimal storage temperature. Biopreservation and Biobanking, 12(3), pp.165–175. doi:10.1089/bio.2013.0084.

24. Doster, W., et al., 1989. Dynamical transition of myoglobin revealed by inelastic neutron scattering. Nature, 337, pp.754–756. doi:10.1038/337754a0.

25. Leonard, S., et al., 1993. Biological stability of mRNA isolated from human postmortem brain collections. Biological Psychiatry, 33(6), pp.456–466. doi:10.1016/0006-3223(93)90174-C.

26. Shabihkhani, M., et al., 2014. The procurement, storage, and quality assurance of frozen blood and tissue biospecimens in pathology, biorepository, and biobank settings. Clinical Biochemistry, 47(4–5), pp.258–266. doi:10.1016/j.clinbiochem.2014.01.002.

27. Kypraiou, C., et al., 2025. Evolution and evaluation of ultra-low temperature freezers: A comprehensive literature review. Foods, 14(13), 2298. doi:10.3390/foods14132298.

28. Muller, R., et al., 2016. Preservation of biospecimens at ambient temperature: Special focus on nucleic acids and opportunities for the biobanking community. Biopreservation and Biobanking, 14(2), pp.89–98.

29. Narvaez Villarrubia, C.W., et al., 2022. Long-term stabilization of DNA at room temperature using a one-step microwave assisted process. Emergent Materials, 5(2), pp.307–314. doi:10.1007/s42247-021-00208-3.

30. Lou, J.J., et al., 2014. A review of room temperature storage of biospecimen tissue and nucleic acids for anatomic pathology laboratories and biorepositories. Clinical Biochemistry, 47(4–5), pp.267–273. doi:10.1016/j.clinbiochem.2013.12.011.

If you have a wish or a question, feel free to reply to this Email.

Otherwise, wish you a beautiful week!
See you again on the 4th : )

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Edited by Patrick Penndorf
Connection@ReAdvance.com
Lutherstraße 159, 07743, Jena, Thuringia, Germany
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Green Education – Is -70 Safe For Your Samples?