Cryopreservation is the gold-standard method to preserve nucleic acids. Typically, samples are snap frozen as soon as possible after collection and then stored, either in −80°C freezers or using a liquid nitrogen (−196°C) system. The preferred storage system is the liquid nitrogen system, because it stores biospecimens below the glass transition point (approximately −135°C). However, it is often not as practical; therefore, storage regularly occurs in −80°C freezers, where samples slowly degrade because of low-level, long-term enzymatic activity. Mathieson et al. (2016) used the Chernobyl Tissue Bank (CTB) to establish whether RNA degrades in long-term storage at −80°C, and whether RNA integrity numbers (RINs) determine fitness for purpose in severely degraded RNA.1
In 1998, following the Chernobyl nuclear accident, there was an increase in thyroid cancers in people who were children at the time of the accident. This was the catalyst for the CTB being established. It is a significant resource for researchers interested in radiation-induced cancer. However, the CTB’s samples are stored at −80°C.
Mathieson et al. analyzed the RIN data from 549 CTB RNA samples to establish whether the RNA had degraded in the tissue samples during their 11-year storage. They extracted RNA using an RNEasy Mini Kit, after homogenizing the tissues in a TissueLyser, and then quantified it using spectrophotometry. Mathieson et al. performed extraction in batches of 11 specimens at one time. They subsequently reverse transcribed the RNA samples to 20 μL using random hexamer primers and a High-Capacity cDNA Reverse Transcription Kit (Thermo Scientific). They followed this with 35 cycles of polymerase chain reaction (PCR) and separation using agarose gel electrophoresis.
The researchers found that sample storage times varied between 0.1 and 10.9 years, and the median RIN for each full year of storage was 7.3–8.2. The 549 samples they used had not undergone a freeze–thaw cycle. PCR returned amplimers of 942 base pairs in samples with a RIN > 8.0 and in 87% of the samples with RINs 5–7.
Mathieson et al. conclude that after 11 years, the CTB’s thyroid biospecimens did not degrade, despite their being stored at temperatures above the glass transition point.
1. Mathieson, W., et al. (2016) “The effect of long-term −80°C storage of thyroid biospecimens on RNA quality and ensuring fitness for purpose,” Journal of Clinical Pathology [Epub ahead of print].