The Effects of Non-Ideal Temperature Regimes on RNA Quality from Samples Stored in RNAlater-like Buffer: An Attempt to Replicate Field Conditions

Advances in sequencing technologies have increased the utility of RNA to many fields of biology. Ideal preservation of RNA in tissues is accomplished by flash freezing and subsequent storage at -80 °C. Buffers such as RNAlater® are commonly used to preserve RNA samples when flash freezing is not possible. RNAlater® performs optimally at subfreezing temperatures, but is also suitable for short periods at room temperature (21 °C). The effectiveness of RNAlater® when samples are stored above room temperature has not been tested. Samples collected outside of the lab are often subjected to temperatures that can exceed room temperature and fluctuate over time. Insect tissues were submerged in RNAlater-like buffer, a noncommercial RNA preservation buffer similar to commercial RNAlater, under various temperature regimes for 21 days. Tests were designed to mimic condition for tissues collected in the field and included constant temperatures of -20 °C, 0 °C, 4 °C, 21 °C, 30 °C, and temperatures that oscillated between -20 °C and 30 °C among others. RNA quality and degradation were determined by electropherograms and electrophoretic gels. Samples yielded high-quality RNA when stored in frozen (-20 °C) or cool (0 °C or 4 °C) conditions, although slightly higher RNA quality was recovered from samples stored in the 0-4 °C temperature range. RNA from samples stored at 21 °C or at oscillating temperatures of -20 °C and 30 °C were of lower quality and were unacceptable for most next-generation sequencing assays. Room temperature appears to be the critical temperature threshold above which RNA in tissues degrades rapidly. Freeze/thaw cycling was shown to mildly improve RNA quality compared to samples stored at a constant 30 °C.