ArticleMethods in molecular biology (Clifton, N.J.)2025
Isolation of High-Quality RNA from Mammalian Spermatozoa for Transcriptome Studies.
Article in Methods in molecular biology (Clifton, N.J.), 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
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.
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