175 Comparison of RNA extraction methods for equine spermatozoal transcriptomics
M. F. Orsolini A , M. van Heule A C , P. Dini A and S. Meyers BA Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, Davis, CA, USA
B Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California, Davis, Davis, CA, USA
C Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium
Reproduction, Fertility and Development 35(2) 215-215 https://doi.org/10.1071/RDv35n2Ab175
Published: 5 December 2022
© 2023 The Author(s) (or their employer(s)). Published by CSIRO Publishing on behalf of the IETS
Sperm RNAs are historically difficult to recover, contributing to the conventional thought that sperm are transcriptionally silent and may not carry functional RNAs. However, recent studies in humans have not only successfully extracted sperm RNAs but also used them for high quality RNA sequencing, leading to new insights into mechanisms of male fertility. In stallions, sperm RNAs have been investigated; however, the utilised methods did not account for contaminants, such as epithelial cells, present in semen. Therefore, in this study, three methods of RNA extraction and preliminary RNA-sequencing data were compared. All extraction methods were performed on sperm samples that were diluted to 20, 40, and 60 million cells per mL. First, density gradient centrifugation was performed to remove somatic cell contaminants followed by either fatty acid RNA purification kit (Norgen, Thorald; FA method), or QIAzol® lysis (QIAzol® Lysis Reagent, Qiagen) supplemented with tris carboxylethatris(2-carboxyethyl) phosphine, followed by an RNeasy column extraction (Qiagen; TCEP method) to extract total RNA from semen. The third method utilised a triple wash rather than a density gradient, followed by a Qiazol phase separation protocol (Qiazol method) to extract total RNA. RNA quantity and quality were measured using a Tapestation 4200 (Agilent Technologies). RNA quality after all three extractions appeared low and RNA was highly fragmented, which aligns with studies in human sperm in which small RNAs (small, non-coding RNAs, microRNAs, tRNA-derived small RNAs) are most abundant. Libraries were prepared from the total RNA using SMART-Seq™ v3 Ultra Low Input RNA Kit. Sequencing was performed on a HiSEqn 4000 platform, generating 12 GB per sample (150PE), and gene ontology was performed using Panther. Sequencing results of the FA RNA showed that % ≥ Q30 reads ranged from 90.24–91.51 across three samples. The tested sperm concentrations played no significant role in final RNA or sequencing quality. TCEP-extracted samples yielded % ≥ Q30 of 93.43–93.83 across three samples. Qiazol-extracted RNA was unable to be successfully amplified. In total, 2,510 genes were expressed in all the samples with 2,393 protein coding genes. Gene expression was not compared among sample groups, as this preliminary project only aimed to characterise the efficacy of sperm RNA extraction methods for downstream analysis. The most abundant pathways associated with the expressed genes were cytoskeleton, microtubule cytoskeleton, RNA binding, enzyme binding, microtubule-based process, and chromatin organisation. We were able to successfully extract RNA from stallion sperm and show that the RNA was sufficient for further downstream analysis such as RNA-seq. It is notable that the TCEP and FA methods, as opposed to the Qiazol method, both utilise supplementary steps to lyse the sperm plasma membrane, potentially improving the total yield of sperm RNA transcripts that are either embedded within or lie beneath the membrane. Future transcriptional studies in stallion sperm should utilise superior methods of RNA extraction, which will subsequently optimise downstream analysis and lead to new insights in stallion fertility and mechanisms of sperm function.
