{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T03:24:56Z","timestamp":1790652296267,"version":"4.1.0"},"reference-count":50,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2021,4,21]],"date-time":"2021-04-21T00:00:00Z","timestamp":1618963200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100006447","name":"Universit\u00e4t Z\u00fcrich","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100006447","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003006","name":"Eidgen\u00f6ssische Technische Hochschule Z\u00fcrich","doi-asserted-by":"publisher","award":["ETH-10 15-2"],"award-info":[{"award-number":["ETH-10 15-2"]}],"id":[{"id":"10.13039\/501100003006","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001711","name":"Schweizerischer Nationalfonds zur F\u00f6rderung der Wissenschaftlichen Forschung","doi-asserted-by":"publisher","award":["31003A_175742\/1"],"award-info":[{"award-number":["31003A_175742\/1"]}],"id":[{"id":"10.13039\/501100001711","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Cell Dev. Biol."],"abstract":"<jats:p>The extraction of high-quality ribonucleic acid (RNA) from tissues and cells is a key step in many biological assays. Guanidinium thiocyanate-phenol-chloroform (AGPC) is a widely used and efficient method to obtain pure RNA from most tissues and cells. However, it is not efficient with some cells like sperm cells because they are resistant to chaotropic lysis solutions containing guanidinium thiocyanate such as Buffer RLT+ and Trizol. Here, we show that disulfide bonds are responsible for the chemical resistance of sperm cells to RNA extraction reagents. We show that while \u03b2-mercaptoethanol (\u03b2ME) can increase sperm lysis in Buffer RLT+, it has no effect in Trizol and leaves sperm cells intact. We measured the reduction of disulfide bonds in 2,2\u2032-dithiodipyridine (DTDP) and observed that \u03b2ME has a pH-dependent activity in chaotropic solutions, suggesting that pH is a limiting factor. We identified tris(2-carboxyethyl)phosphine (TCEP) as an efficient lysis enhancer of AGPC solutions that can retain reducing activity even at acidic pH. Trizol supplemented with TCEP allows the complete and rapid lysis of sperm cells, increasing RNA yield by 100-fold and resulting in RNA with optimal quality for reverse transcription and polymerase chain reaction. Our findings highlight the importance of efficient cell lysis and extraction of various macromolecules for bulk and single-cell assays, and can be applied to other lysis-resistant cells and vesicles, thereby optimizing the amount of required starting material and animals.<\/jats:p>","DOI":"10.3389\/fcell.2021.648274","type":"journal-article","created":{"date-parts":[[2021,4,21]],"date-time":"2021-04-21T07:48:56Z","timestamp":1618991336000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":34,"title":["High Efficiency RNA Extraction From Sperm Cells Using Guanidinium Thiocyanate Supplemented With Tris(2-Carboxyethyl)Phosphine"],"prefix":"10.3389","volume":"9","author":[{"given":"Martin","family":"Roszkowski","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Isabelle M.","family":"Mansuy","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2021,4,21]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"178","DOI":"10.1016\/j.chemphyslip.2018.12.008","article-title":"Mechanical properties of bilayers containing sperm sphingomyelins and ceramides with very long-chain polyunsaturated fatty acids.","volume":"218","author":"Ahumada-Gutierrez","year":"2019","journal-title":"Chem. Phys. Lipids"},{"key":"B2","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1155\/2017\/9306564","article-title":"Current nucleic acid extraction methods and their implications to point-of-care diagnostics.","volume":"2017","author":"Ali","year":"2017","journal-title":"Biomed Res. Int."},{"key":"B3","doi-asserted-by":"publisher","first-page":"315","DOI":"10.1105\/tpc.18.00872","article-title":"Plant extracellular vesicles contain diverse small RNA species and are enriched in 10- to 17-nucleotide \u201cTiny\u201d RNAs.","volume":"31","author":"Baldrich","year":"2019","journal-title":"Plant Cell"},{"key":"B4","doi-asserted-by":"publisher","first-page":"2056","DOI":"10.1016\/S0006-3495(96)79404-3","article-title":"How Hofmeister ion interactions affect protein stability.","volume":"71","author":"Baldwin","year":"1996","journal-title":"Biophys. J."},{"key":"B5","doi-asserted-by":"publisher","first-page":"374","DOI":"10.1111\/andr.12471","article-title":"High-quality human and rat spermatozoal RNA isolation for functional genomic studies.","volume":"6","author":"Bianchi","year":"2018","journal-title":"Andrology"},{"key":"B6","doi-asserted-by":"publisher","first-page":"641","DOI":"10.1038\/nrg3964","article-title":"Molecular insights into transgenerational non-genetic inheritance of acquired behaviours.","volume":"16","author":"Bohacek","year":"2015","journal-title":"Nat. Rev. Genet."},{"key":"B7","doi-asserted-by":"publisher","first-page":"495","DOI":"10.1128\/JCM.28.3.495-503.1990","article-title":"Rapid and simple method for purification of nucleic acids.","volume":"28","author":"Boom","year":"1990","journal-title":"J. Clin. Microbiol."},{"key":"B8","doi-asserted-by":"publisher","first-page":"630","DOI":"10.1038\/s41576-020-0258-4","article-title":"mRNAs, proteins and the emerging principles of gene expression control.","volume":"21","author":"Buccitelli","year":"2020","journal-title":"Nat. Rev. Genet"},{"key":"B9","doi-asserted-by":"publisher","first-page":"611","DOI":"10.1373\/clinchem.2008.112797","article-title":"The MIQE guidelines:minimum information for publication of quantitative real-time PCR experiments.","volume":"55","author":"Bustin","year":"2009","journal-title":"Clin. Chem."},{"key":"B10","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0035538","article-title":"Mouse estrous cycle identification tool and images.","volume":"7","author":"Byers","year":"2012","journal-title":"PLoS One"},{"key":"B11","doi-asserted-by":"publisher","first-page":"73","DOI":"10.1016\/S0014-5793(01)03284-7","article-title":"The unfolding mechanism and the disulfide structures of denatured lysozyme.","volume":"511","author":"Chang","year":"2002","journal-title":"FEBS Lett."},{"key":"B12","doi-asserted-by":"publisher","first-page":"156","DOI":"10.1016\/0003-2697(87)90021-2","article-title":"Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.","volume":"162","author":"Chomczynski","year":"1987","journal-title":"Anal. Biochem."},{"key":"B13","doi-asserted-by":"publisher","first-page":"581","DOI":"10.1038\/nprot.2006.83","article-title":"The single-step method of RNA isolation by acid guanidinium thiocyanate\u2013phenol\u2013chloroform extraction: twenty-something years on.","volume":"1","author":"Chomczynski","year":"2006","journal-title":"Nat. Protoc."},{"key":"B14","doi-asserted-by":"publisher","first-page":"1379","DOI":"10.1007\/s10815-019-01464-3","article-title":"Decreased very long chain polyunsaturated fatty acids in sperm correlates with sperm quantity and quality.","volume":"36","author":"Craig","year":"2019","journal-title":"J. Assist. Reprod. Genet."},{"key":"B15","doi-asserted-by":"publisher","first-page":"231","DOI":"10.1002\/mrd.1120080304","article-title":"The sperm head of the plains mouse, Pseudomys australis: ultrastructure and effects of chemical treatments.","volume":"8","author":"Flaherty","year":"1983","journal-title":"Gamete Res."},{"key":"B16","first-page":"404","article-title":"Phase dependent discrepancy in murine vaginal micro-environment: a correlative analysis of PH, glycogen and serum estrogen upon exposure to lapatinib ditosylate.","volume":"8","author":"Ganesan","year":"2016","journal-title":"Int. J. Pharm. Pharm. Sci."},{"key":"B17","doi-asserted-by":"publisher","first-page":"1073","DOI":"10.1530\/REP-06-0292","article-title":"A molecular analysis of the population of mRNA in bovine spermatozoa.","volume":"133","author":"Gilbert","year":"2007","journal-title":"Reproduction"},{"key":"B18","doi-asserted-by":"publisher","first-page":"5","DOI":"10.1006\/abio.1994.1290","article-title":"A procedure for quantitative determination of Tris(2-Carboxyethyl)phosphine, an odorless reducing agent more stable and effective than dithiothreitol.","volume":"220","author":"Han","year":"1994","journal-title":"Anal. Biochem."},{"key":"B19","doi-asserted-by":"publisher","first-page":"2449","DOI":"10.1038\/nprot.2013.145","article-title":"Genome-wide chromatin analysis in mature mouse and human spermatozoa.","volume":"8","author":"Hisano","year":"2013","journal-title":"Nat. Protoc."},{"key":"B20","doi-asserted-by":"publisher","first-page":"71","DOI":"10.1038\/nrg3863","article-title":"RNA-mediated epigenetic regulation of gene expression.","volume":"16","author":"Holoch","year":"2015","journal-title":"Nat. Rev. Genet."},{"key":"B21","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2164-8-39","article-title":"A screen for nuclear transcripts identifies two linked noncoding RNAs associated with SC35 splicing domains.","volume":"8","author":"Hutchinson","year":"2007","journal-title":"BMC Genomics"},{"key":"B22","doi-asserted-by":"publisher","first-page":"1925","DOI":"10.1016\/j.bpj.2017.08.050","article-title":"Role of disulfide bonds on DNA packaging forces in bull sperm chromatin.","volume":"113","author":"Hutchison","year":"2017","journal-title":"Biophys. J."},{"key":"B23","doi-asserted-by":"publisher","first-page":"1366","DOI":"10.1016\/j.celrep.2017.01.034","article-title":"Chromatin states in mouse sperm correlate with embryonic and adult regulatory landscapes.","volume":"18","author":"Jung","year":"2017","journal-title":"Cell Rep."},{"key":"B24","doi-asserted-by":"publisher","first-page":"893","DOI":"10.1016\/j.molcel.2012.05.036","article-title":"Short structured RNAs with low GC content are selectively lost during extraction from a small number of cells.","volume":"46","author":"Kim","year":"2012","journal-title":"Mol. Cell"},{"key":"B25","doi-asserted-by":"publisher","first-page":"633","DOI":"10.1038\/nrg1654","article-title":"Paternal contribution: new insights and future challenges.","volume":"6","author":"Krawetz","year":"2005","journal-title":"Nat. Rev. Genet."},{"key":"B26","doi-asserted-by":"publisher","first-page":"2467","DOI":"10.1007\/s00216-017-0772-1","article-title":"Recent mass spectrometry-based techniques and considerations for disulfide bond characterization in proteins.","volume":"410","author":"Lakbub","year":"2018","journal-title":"Anal. Bioanal. Chem."},{"key":"B27","doi-asserted-by":"publisher","first-page":"535","DOI":"10.1016\/j.tins.2013.05.004","article-title":"New mechanisms in memory storage: piRNAs and epigenetics.","volume":"36","author":"Landry","year":"2013","journal-title":"Trends Neurosci."},{"key":"B28","doi-asserted-by":"publisher","first-page":"11380","DOI":"10.1093\/nar\/gkaa712","article-title":"Sperm histone H3 lysine 4 trimethylation is altered in a genetic mouse model of transgenerational epigenetic inheritance.","volume":"48","author":"Lismer","year":"2020","journal-title":"Nucleic Acids Res."},{"key":"B29","doi-asserted-by":"publisher","first-page":"519","DOI":"10.1038\/nmeth.3370","article-title":"G&T-seq: parallel sequencing of single-cell genomes and transcriptomes.","volume":"12","author":"Macaulay","year":"2015","journal-title":"Nat. Methods"},{"key":"B30","doi-asserted-by":"publisher","first-page":"4557","DOI":"10.1073\/pnas.0735920100","article-title":"The hydration structure of guanidinium and thiocyanate ions: implications for protein stability in aqueous solution.","volume":"100","author":"Mason","year":"2003","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"B31","doi-asserted-by":"publisher","first-page":"174","DOI":"10.1038\/nature00832","article-title":"Exceptional sperm cooperation in the wood mouse.","volume":"418","author":"Moore","year":"2002","journal-title":"Nature"},{"key":"B32","doi-asserted-by":"publisher","first-page":"1149","DOI":"10.1016\/j.bbrc.2014.05.100","article-title":"Intracellular pH in sperm physiology.","volume":"450","author":"Nishigaki","year":"2014","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"B33","doi-asserted-by":"publisher","first-page":"585","DOI":"10.1038\/s41580-020-0251-y","article-title":"RNA delivery by extracellular vesicles in mammalian cells and its applications.","volume":"21","author":"O\u2019Brien","year":"2020","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"B34","doi-asserted-by":"publisher","first-page":"89","DOI":"10.1038\/s41576-018-0073-3","article-title":"PIWI-interacting RNAs: small RNAs with big functions.","volume":"20","author":"Ozata","year":"2019","journal-title":"Nat. Rev. Genet."},{"key":"B35","doi-asserted-by":"publisher","first-page":"1609","DOI":"10.1038\/cr.2012.141","article-title":"A novel class of tRNA-derived small RNAs extremely enriched in mature mouse sperm.","volume":"22","author":"Peng","year":"2012","journal-title":"Cell Res."},{"key":"B36","doi-asserted-by":"publisher","first-page":"272","DOI":"10.1016\/S0006-291X(89)80208-6","article-title":"Presence of RNA in the sperm nucleus.","volume":"158","author":"Pessot","year":"1989","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"B37","doi-asserted-by":"publisher","first-page":"47","DOI":"10.1038\/nrg.2015.10","article-title":"Unique features of long non-coding RNA biogenesis and function.","volume":"17","author":"Quinn","year":"2016","journal-title":"Nat. Rev. Genet."},{"key":"B38","doi-asserted-by":"publisher","first-page":"R720","DOI":"10.1016\/j.cub.2017.05.043","article-title":"Principles of transgenerational small RNA inheritance in Caenorhabditis elegans.","volume":"27","author":"Rechavi","year":"2017","journal-title":"Curr. Biol."},{"key":"B39","doi-asserted-by":"publisher","first-page":"137","DOI":"10.1016\/j.ab.2003.10.019","article-title":"Tris(2-carboxyethyl)phosphine stabilization of RNA: comparison with dithiothreitol for use with nucleic acid and thiophosphoryl chemistry.","volume":"325","author":"Rhee","year":"2004","journal-title":"Anal. Biochem."},{"key":"B40","doi-asserted-by":"publisher","first-page":"2935","DOI":"10.1073\/pnas.1808696116","article-title":"Molecular basis for AU-rich element recognition and dimerization by the HuR C-terminal RRM.","volume":"116","author":"Ripin","year":"2019","journal-title":"Proc. Natl. Acad. Sci.U.S.A."},{"key":"B41","doi-asserted-by":"crossref","first-page":"1746","DOI":"10.1016\/S0021-9258(18)46009-7","article-title":"Novel molecular species of sphingomyelin containing 2-hydroxylated polyenoic very-long-chain fatty acids in mammalian testes and spermatozoa.","volume":"267","author":"Robinson","year":"1992","journal-title":"J. Biol. Chem."},{"key":"B42","doi-asserted-by":"publisher","first-page":"382","DOI":"10.1046\/j.1537-2995.2001.41030382.x","article-title":"Virus inactivation of plasma-derived proteins by pasteurization in the presence of guanidine hydrochloride.","volume":"41","author":"Schlegel","year":"2001","journal-title":"Transfusion"},{"key":"B43","doi-asserted-by":"publisher","first-page":"259","DOI":"10.1016\/j.anireprosci.2014.06.032","article-title":"Development of a new method for sperm RNA purification in the chicken.","volume":"149","author":"Shafeeque","year":"2014","journal-title":"Anim. Reprod. Sci."},{"key":"B44","doi-asserted-by":"publisher","first-page":"631","DOI":"10.1038\/s41576-019-0150-2","article-title":"RNA sequencing: the teenage years.","volume":"20","author":"Stark","year":"2019","journal-title":"Nat. Rev. Genet."},{"key":"B45","doi-asserted-by":"publisher","first-page":"834","DOI":"10.3892\/ijmm.2017.3080","article-title":"Comparison of isolation methods of exosomes and exosomal RNA from cell culture medium and serum.","volume":"40","author":"Tang","year":"2017","journal-title":"Int. J. Mol. Med."},{"key":"B46","doi-asserted-by":"publisher","DOI":"10.1016\/j.isci.2020.101079","article-title":"Orthogonal proteomic platforms and their implications for the stable classification of high-grade serous ovarian cancer subtypes.","volume":"23","author":"Thomas","year":"2020","journal-title":"iScience"},{"key":"B47","doi-asserted-by":"publisher","first-page":"599","DOI":"10.1016\/j.mex.2018.05.011","article-title":"Optimization of phenol-chloroform RNA extraction.","volume":"5","author":"Toni","year":"2018","journal-title":"MethodsX"},{"key":"B48","doi-asserted-by":"publisher","first-page":"3920","DOI":"10.1016\/j.celrep.2018.05.094","article-title":"Re-evaluating the localization of sperm-retained histones revealed the modification-dependent accumulation in specific genome regions.","volume":"23","author":"Yamaguchi","year":"2018","journal-title":"Cell Rep."},{"key":"B49","doi-asserted-by":"publisher","first-page":"1778","DOI":"10.1016\/j.biochi.2010.08.012","article-title":"Ceramides with 2-hydroxylated, very long-chain polyenoic fatty acids in rodents: from testis to fertilization-competent spermatozoa.","volume":"92","author":"Zanetti","year":"2010","journal-title":"Biochimie"},{"key":"B50","doi-asserted-by":"publisher","first-page":"91","DOI":"10.1016\/B978-0-12-622595-2.50011-5","article-title":"Formation and function of the male pronucleus during mammalian fertilization.","author":"Zirkin","year":"1989","journal-title":"Mol. Biol. Fertili."}],"container-title":["Frontiers in Cell and Developmental Biology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fcell.2021.648274\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,4,21]],"date-time":"2021-04-21T07:49:01Z","timestamp":1618991341000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fcell.2021.648274\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,4,21]]},"references-count":50,"alternative-id":["10.3389\/fcell.2021.648274"],"URL":"https:\/\/doi.org\/10.3389\/fcell.2021.648274","relation":{},"ISSN":["2296-634X"],"issn-type":[{"value":"2296-634X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,4,21]]},"article-number":"648274"}}