Isolation of High-Quality RNA from Mammalian Spermatozoa for Transcriptome Studies

Mammalian spermatozoa contain a suite of RNA, including mRNA and miRNAs, that may serve as molecular markers of male fertility. The whole transcriptome analysis also identified a few full-length mRNA transcripts in mammalian spermatozoa. However, the isolation of spermatozoal RNA (spRNA) is challenging due to its extremely low concentration and highly condensed nucleo-protamine complex in spermatozoa. Further, chemical agents used for the lysis of cells decrease the total yield and quality of RNAs. Here, we provide an optimized protocol for extracting high-quality spRNA suitable for both short-read (e.g., Illumina™ sequencing) and long-read (e.g., Nanopore™ sequencing) RNA sequencing (RNA-seq). The protocol involves the treatment of semen samples with a lysis solution to remove somatic cells, followed by treatment with a monophasic solution of guanidium thiocyanate (GITC) and 1,4-Dithiothreitol (DTT) to obtain a high-yield of high-quality spRNA.