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Total RNA isolation from stallion sperm and testis biopsies.
Abstract: Sperm mRNA transcriptional profiles can be used to evaluate male fertility, yet differences between species in sperm attributes and packaging require adjustments in sperm RNA isolation protocols. The objective was to optimize RNA isolation methodology for fresh, frozen, and extended ejaculates, and epididymal sperm of stallions. Additionally, a protocol for RNA isolation from testis biopsies was established. Separation of sperm from somatic cells was critical for assuring the isolation of sperm-specific RNA. The highest purity was obtained by centrifuging ejaculates and epididymal sperm at 200 x g for 30 min through a 40% Equipure silanized silica particle solution. Sperm RNA isolation was more efficient with TRIzol reagent than with a spin-column based method; it resulted in 2 microg of total RNA per 100 x 10(6) sperm. To evaluate RNA quantity and quality, we used a NanoDrop spectrophotometer and Agilent Bioanalyzer. A protocol for reverse transcriptase PCR with equine primers for PRM2 and PTPRC genes was developed to determine sperm RNA contamination with genomic DNA or RNA from somatic cells. By these methods, hybridization- and sequencing-quality RNA was isolated from 11 samples of stallion sperm. Stallion testis biopsy with a 14 gauge 22 mm deep biopsy needle yielded approximately 12 microg of good quality total RNA, and could serve as an alternative to excision surgery for sample procurement. Compared to RNA isolation from testis, the sperm required advanced processing and RNA quality control. The described methodologies provided a foundation to establish functional genomic studies of stallion fertility.
(c) 2010 Elsevier Inc. All rights reserved.
Publication Date: 2010-07-07 PubMed ID: 20615536DOI: 10.1016/j.theriogenology.2010.04.023Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
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Summary
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This research focused on developing an effective method to isolate RNA from stallion sperm and testis biopsies. The goal was to enhance the evaluation of male fertility by examining the mRNA transcriptional profiles present in sperm. Specific protocols were optimized for different types of ejaculates and even a new protocol for RNA isolation from testis biopsies was established.
Optimizing RNA Isolation
- The scientists aimed to improve RNA isolation methodologies for different kinds of sperm samples, namely fresh, frozen, extended ejaculates and epididymal sperm. This process was critical in ensuring only sperm-specific RNA was isolated, a significant requirement when evaluating male fertility as it offers insights into the functional state of sperm cells.
- The optimal method found was by centrifuging the ejaculates and epididymal sperm through a silanized silica particle solution. This ensured the highest purity and separation of sperm from other somatic cells.
- Among the isolation methods used, TRIzol reagent proved more efficient than the spin-column based method. This yielded around 2 micrograms of total RNA per 100 million sperm.
Evaluating RNA Quantity and Quality
- To ensure the RNA was of high quality and sufficient quantity for subsequent analysis, the researchers used a NanoDrop spectrophotometer and an Agilent Bioanalyzer. These devices allowed precise quantification and quality assessment of the isolated RNA.
- A specific protocol for reverse transcriptase PCR with equine primers for PRM2 and PTPRC genes was developed. This helped ascertain if the sperm RNA was contaminated with either genomic DNA or RNA from somatic cells.
- Taking all these measures, the researchers were able to isolate high-quality RNA from 11 samples of stallion sperm suitable for hybridization and sequencing.
RNA Isolation from Testis Biopsies
- Along with the sperm samples, this study also established a protocol for isolating RNA from testis biopsies. A 14 gauge 22 mm deep biopsy needle was applied to obtain around 12 micrograms of good quality total RNA. This technique serves as a less invasive method compared to traditional excision surgeries for sample collection.
- However, compared to testis RNA isolation, the isolation process from sperm required advanced processing and more stringent quality control. This emphasizes the delicate and complex nature of sperm samples.
Implications for Stallion Fertility Studies
- The methods established in this research paper provide a solid base for future functional genomic studies of stallion fertility. Implementing these optimized procedures will ensure separation of sperm-specific RNA and minimize contamination.
- Ultimately, this would assist in more accurate assessment of male fertility through examining mRNA transcriptional profiles, offering potential insights into fertility issues and improving breeding strategies.
Cite This Article
APA
Das PJ, Paria N, Gustafson-Seabury A, Vishnoi M, Chaki SP, Love CC, Varner DD, Chowdhary BP, Raudsepp T.
(2010).
Total RNA isolation from stallion sperm and testis biopsies.
Theriogenology, 74(6), 1099-1106e11062.
https://doi.org/10.1016/j.theriogenology.2010.04.023 Publication
Researcher Affiliations
- Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX77843, USA.
MeSH Terms
- Animals
- Biopsy / veterinary
- Cryopreservation / methods
- Cryopreservation / veterinary
- Genetic Techniques
- Horses / genetics
- Horses / metabolism
- Male
- Quality Control
- RNA / analysis
- RNA / isolation & purification
- RNA / metabolism
- Reverse Transcriptase Polymerase Chain Reaction
- Semen Analysis
- Semen Preservation / methods
- Semen Preservation / veterinary
- Spermatozoa / chemistry
- Spermatozoa / metabolism
- Testis / chemistry
- Testis / pathology
Citations
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