Our modular sperm RNA isolation protocol uses the mirVana miRNA Isolation Kit (Ambion), with slight modifications at the lysis stage (the ‘Modular Step’). The Modular Step is followed by addition of the miRNA homogenate additive and the rest of the standard total RNA isolation protocol. Due to differences in sperm morphology between species, this Modular Step needs to be tailored to each species. Samples should be free of non-sperm cell contamination – somatic cells contain ~250 times more RNA than sperm do, so a little contamination can heavily skew results! Sperm RNA are also notoriously susceptible to degradation, therefore, make sure to test our method (and optimize if necessary) for your own samples before working with precious samples.
Conventional RNA sample quality metrics should not be applied to sperm RNA – sperm-borne RNA are fragmented and are deficient in intact 18S and 28S rRNA. However, the RNA integrity number (RIN) is still a useful measurement for assessing sperm RNA quality. From our experience, high quality sperm RNA samples have a RIN of ~3; a RIN < 2 indicates excessive sample degradation and a RIN > 4 indicates somatic cell contamination.