Research Article

Evaluation of the semen swim-up method for bovine sperm RNA extraction

Published: May 06, 2016
Genet. Mol. Res. 15(2): gmr7713 DOI: https://doi.org/10.4238/gmr.15027713
Cite this Article:
(2016). Evaluation of the semen swim-up method for bovine sperm RNA extraction. Genet. Mol. Res. 15(2): gmr7713. https://doi.org/10.4238/gmr.15027713
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Abstract

Isolation of high-quality RNA is important for assessing sperm gene expression, and semen purification methods may affect the integrity of the isolated RNA. This study evaluated the effectiveness of the sperm swim-up method for seminal RNA isolation. Frozen semen samples in straws from three bulls of proven fertility were purified by the swim-up method. RNA extraction was carried out using the E.Z.N.A.TM Total RNA kit II, with non-swim-up sperm as a control. Total sperm RNA was analyzed by UV spectrophotometry, reverse transcription polymerase chain reaction (RT-PCR), and agarose gel electrophoresis, and expression of the sex-determining region on the Y chromosome (SRY), leptin (LEP), and ribosomal protein subunit 23 (RPS23) genes, were determined. 18S RNA was used as a positive control. Fewer somatic cells were found in sperm swim-up samples than in the non-swim-up counterparts (0 x 103 vs 17.33 ± 2.52 x 103 sperm, P 7 sperm) compared with the non-swim-up samples (1.36 ± 0.33 x 107 sperm, P SRY, LEP, and RPS23 in sperm cDNA were observed on agarose gel electrophoresis. Finally, no bands corresponding to 18S RNA were found in RNA samples from the sperm swim-up group. Our findings suggest that small amounts of sperm RNA can be efficiently extracted from frozen straw semen samples using the swim-up technique.

Isolation of high-quality RNA is important for assessing sperm gene expression, and semen purification methods may affect the integrity of the isolated RNA. This study evaluated the effectiveness of the sperm swim-up method for seminal RNA isolation. Frozen semen samples in straws from three bulls of proven fertility were purified by the swim-up method. RNA extraction was carried out using the E.Z.N.A.TM Total RNA kit II, with non-swim-up sperm as a control. Total sperm RNA was analyzed by UV spectrophotometry, reverse transcription polymerase chain reaction (RT-PCR), and agarose gel electrophoresis, and expression of the sex-determining region on the Y chromosome (SRY), leptin (LEP), and ribosomal protein subunit 23 (RPS23) genes, were determined. 18S RNA was used as a positive control. Fewer somatic cells were found in sperm swim-up samples than in the non-swim-up counterparts (0 x 103 vs 17.33 ± 2.52 x 103 sperm, P 7 sperm) compared with the non-swim-up samples (1.36 ± 0.33 x 107 sperm, P SRY, LEP, and RPS23 in sperm cDNA were observed on agarose gel electrophoresis. Finally, no bands corresponding to 18S RNA were found in RNA samples from the sperm swim-up group. Our findings suggest that small amounts of sperm RNA can be efficiently extracted from frozen straw semen samples using the swim-up technique.