ArticleFrontiers in cell and developmental biology2021
High Efficiency RNA Extraction From Sperm Cells Using Guanidinium Thiocyanate Supplemented With Tris(2-Carboxyethyl)Phosphine.
Article in Frontiers in cell and developmental biology, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 14 papers.
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14 citing papers in PubMed, 28 citations in OpenAlex.
- Testicular toll with viral intrusion: Molecular insights into the impact of chronic HIV infection on male fertility.Clinical and translational medicine · 2026Article
- Sperm TLR2/1 activation modulates the paternal RNA cargo and enhances bovine embryonic development.Frontiers in physiology · 2026Article
- Development and Optimization of an RNA-Isolating Protocol for Mammalian Spermatozoa.International journal of molecular sciences · 2025Article
- Proteomic Analysis of Plant-Derived hIGF-1-Fc Reveals Proteome Abundance Changes Associated with Wound Healing and Cell Proliferation.Proteomes · 2025Article
- Small RNA in sperm-Paternal contributions to human embryo development.Nature communications · 2025Article
- Copy number determination of sperm-borne small RNAs implied in the intergenerational inheritance of metabolic syndromes.RNA (New York, N.Y.) · 2025Article
- Development and optimization of an efficient RNA isolation protocol from bovine (Biochemistry and biophysics reports · 2024Article
- Enhanced detection of RNA modifications and read mapping with high-accuracy nanopore RNA basecalling models.Genome research · 2024Article
- Male infertility is associated with differential DNA methylation signatures of the imprinted gene GNAS and the non-imprinted gene CEP41.Journal of assisted reproduction and genetics · 2024Article
- Alterations in sperm RNAs persist after alcohol cessation and correlate with epididymal mitochondrial dysfunction.Andrology · 2024Article
- Article
- Effectiveness of TRIzol in Inactivating Animal Pathogens.Applied biosafety : journal of the American Biological Safety Association · 2023Review
- Sperm Transcriptome Analysis Accurately Reveals Male Fertility Potential in Livestock.Animals : an open access journal from MDPI · 2022Review
- Bta-miR-6531 Regulates Calcium Influx in Bovine Leydig Cells and Is Associated with Sperm Motility.Genes · 2022Article
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Authors and funding
2 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
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 β-mercaptoethanol (βME) 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'-dithiodipyridine (DTDP) and observed that βME 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.
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