RNA quality and protamine gene expression after storage of mouse testes under different conditions

Protamines are proteins responsible for condensing sperm chromatin. There are two protamines whose ratio remains constant in each species and which is related to fertility. To quantify their expression, it is necessary to have a good protocol of sample collection (i.e., RNA stabilizing buffers and temperature conditions). The aim of this work was to compare gene expression of protamines, with analysis of RNA quality and ratios, in testis samples from wild-derived mice, Mus musculus, preserved in different buffers (RNAlater® or Nucleic Acid Preservation-NAP-buffer) and different temperatures (room temperature -RT-, 4°C, -20°C, -80°C or liquid nitrogen) for different times (one week, one month, 3 months and one year). The relative abundance of protamine expression was assessed by qPCR using 18S rRNA as housekeeping. The results showed that the preservation of testes in RNAlater® or NAP buffer at -80°C afforded equivalent good preservation as in somatic tissues. Testis samples stored at RT in both buffers for 1 week resulted in a similar RNA quality and protamine expression over time. Moreover, samples in RNAlater® stored at RT, 4°C, -20°C and -80°C, were analyzed after 24 h, 7 days, 30 days, 90 days or 365 days; samples stored at RT resulted in a loss of RNA quality but protamine ratio was maintained up to 90 days. Samples stored at 4°C and -20°C showed similar values of RNA integrity and protamine expression than those stored at -80°C. Finally, we stored testis samples at -80°C or -196°C, after initial snap-freezing in liquid nitrogen. Both methods afforded very good preservation of RNA integrity and protamine expression. These results open new possibilities for the collection, transport and storage of testes samples under field conditions.