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:
C.M. Han, R. Chen, T. Li, X.L. Chen, Y.F. Zheng, M.T. Ma, Q.H. Gao, C.M. Han, R. Chen, T. Li, X.L. Chen, Y.F. Zheng, M.T. Ma, Q.H. Gao (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 < 0.05). In addition, high-quality RNA was obtained in about 2 h, with no significant difference between groups. Interestingly, the yields of RNA fragments containing ≥200 nucleotides were significantly reduced in sperm swim-up samples (0.92 ± 0.41 x 107 sperm) compared with the non-swim-up samples (1.36 ± 0.33 x 107 sperm, P < 0.05). After RT-PCR, clear bands representing 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.