Total RNA isolation from stallion sperm and testis biopsies

Pranab J. Das, Nandina Paria, Ashley Gustafson-Seabury, Monika Vishnoi, Sankar P. Chaki, Charles C. Love, Dickson D. Varner, Bhanu P. Chowdhary, Terje Raudsepp

Research output: Contribution to journalArticlepeer-review

59 Scopus citations

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 ×x00D7; g for 30 min through a 40% EquipureTM silanized silica particle solution. Sperm RNA isolation was more efficient with TRIzol reagent than with a spin-column based method; it resulted in 2 μg of total RNA per 100 ×x00D7; 106 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 ~12 μg 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.

Original languageEnglish (US)
Pages (from-to)1099-1106.e2
JournalTheriogenology
Volume74
Issue number6
DOIs
StatePublished - Oct 2010

Keywords

  • Functional genomics
  • RNA
  • Sperm
  • Stallion fertility
  • Testicular biopsy

ASJC Scopus subject areas

  • Small Animals
  • Food Animals
  • Animal Science and Zoology
  • Equine

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