{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,14]],"date-time":"2026-03-14T22:38:31Z","timestamp":1773527911414,"version":"3.50.1"},"reference-count":44,"publisher":"MDPI AG","issue":"22","license":[{"start":{"date-parts":[[2025,11,19]],"date-time":"2025-11-19T00:00:00Z","timestamp":1763510400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"annual recurrent budget of the Universities"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>The RNAs present in spermatozoa play a crucial role in reproduction and embryonic development. They represent a promising diagnostic tool for assessing male infertility. However, their extraction is challenging due to their low concentration and highly condensed chromatin structure, as well as the presence of numerous cellular contaminants. These challenges vary across species and require the development of an optimized and reliable isolation method to obtain high-quality RNAs, which is essential for further molecular analyses regarding the roles played by these RNAs. This study evaluated two RNA extraction methods for spermatozoa in humans and other mammals (dogs, stallions, and bulls): a standard method using the NucleoSpin RNA\u00ae II kit (Macherey-Nagel) and an optimized method that combined this kit with dithiothreitol and TRIzol\u2122 pretreatment. In addition, the samples underwent pre-purification to eliminate somatic cells. The optimized method produced a significantly higher total RNA yield along with better purity, which was confirmed by the absence of the 18S and 28S ribosomal RNA peaks, indicating the absence of somatic cell contamination. Additionally, RT-PCR was performed to validate the presence of sperm-specific markers. The quality of the extracted RNAs was assessed by sequencing the mRNA encoding the human olfactory receptor OR1D2 and observing the resulting band on an agarose slab gel with a size corresponding to its entire open reading frame. By addressing long-standing challenges in sperm RNA isolation, our method provides an easy and standardized technique for research in reproductive biology and for advancing our understanding of paternal contributions to transgenerational inheritance.<\/jats:p>","DOI":"10.3390\/ijms262211171","type":"journal-article","created":{"date-parts":[[2025,11,19]],"date-time":"2025-11-19T11:17:27Z","timestamp":1763551047000},"page":"11171","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Development and Optimization of an RNA-Isolating Protocol for Mammalian Spermatozoa"],"prefix":"10.3390","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0009-0009-2893-6525","authenticated-orcid":false,"given":"El Oulidi","family":"Mounia","sequence":"first","affiliation":[{"name":"Laboratory of Biology and Health, Ibn Tofail University, Kenitra 14000, Morocco"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Azzouzi","family":"Naoual","sequence":"additional","affiliation":[{"name":"Institut de G\u00e9n\u00e9tique et D\u00e9veloppement de Rennes, UMR6290-CNRS, Universit\u00e9 de Rennes, 35000 Rennes, France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5013-6932","authenticated-orcid":false,"given":"Ravel","family":"Celia","sequence":"additional","affiliation":[{"name":"CHU de RENNES-Service de Biologie de la Reproduction-CECOS, Institut National de la Sant\u00e9 et de la Recherche M\u00e9dicale, Ecole des Hautes Etudes en Sant\u00e9 Publique, Institut de Recherche en Sant\u00e9, Environnement et Travail, Universit\u00e9 de Rennes, UMR_S1085, 35000 Rennes, France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5558-7780","authenticated-orcid":false,"given":"Hassani Idrissi","family":"Hind","sequence":"additional","affiliation":[{"name":"Laboratory of Biology and Health, Ibn Tofail University, Kenitra 14000, Morocco"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8060-5661","authenticated-orcid":false,"given":"Habbane","family":"Mouna","sequence":"additional","affiliation":[{"name":"Laboratory of Biology and Health, Ibn Tofail University, Kenitra 14000, Morocco"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fieni","family":"Francis","sequence":"additional","affiliation":[{"name":"Veterinary Reproduction Service ONIRIS of Nantes, 44300 Nantes, France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9596-9815","authenticated-orcid":false,"given":"Galibert","family":"Francis","sequence":"additional","affiliation":[{"name":"Institut de G\u00e9n\u00e9tique et D\u00e9veloppement de Rennes, UMR6290-CNRS, Universit\u00e9 de Rennes, 35000 Rennes, France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5279-491X","authenticated-orcid":false,"given":"Akhouayri","family":"Omar","sequence":"additional","affiliation":[{"name":"Laboratory of Biology and Health, Ibn Tofail University, Kenitra 14000, Morocco"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,11,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"257","DOI":"10.1016\/bs.ctdb.2018.12.006","article-title":"Gametogenesis: A journey from inception to conception","volume":"132","author":"Larose","year":"2019","journal-title":"Curr. 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