Abstract

PEREZ RICO, A. et al. Reference genes selection in cryopreserved equine semen for its use in studies of gene expression with the quantitative PCR technique. Sanid. Mil. [online]. 2014, vol.70, n.1, pp.20-24. ISSN 1887-8571.  https://dx.doi.org/10.4321/S1887-85712014000100003.

Introduction: The germoplasm bank management involves the conservation and use of semen doses, but can also be a source of study on the quality of stallions and semen properties for use after thawing. A criterion for measuring the semen quality may be based on differences in expression of some genes involved in spermatogenesis and sperm maturation. Objective: Analysis of genes expressed in equine cryopreserved sperm that can provide adequate amplification, specificity and stability for use as future reference genes in gene expression studies. Material and methods: Purification of live sperm through a discontinuous concentration gradient from cryopreserved semen straws corresponding to four stallions. Organic extraction of ribonucleic acids with deoxyribonuclease treatment and the selective amplification of seven candidate genes using a retrotranscription and a real time chain reaction of the polymerase in one step mode. Specificity is tested by melting curves and agarose gel electrophoresis. Also the stability of the genes is calculated. Results: Three of the selected genes, α-actin, Ubiquitin B and Ribosomal protein L32 were properly amplified. β-Actin and Ubiquitin B showed the best stability. Conclusion: mRNA was amplified from equine cryopreserved semen samples, being the β-Actin and the Ubiquitin B genes the most suitable reference genes of the seven candidates analyzed.

Keywords : Messenger RNA; Reference genes; Equine cryopreserved sperm.

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