中文摘要：

优选3头公牛细管冻精,采用上游法获得纯化精子,以含体细胞污染的精子作对照,应用离心层析柱方法和Trizol一步法相结合提取总RNA,紫外分光光度计检测总RNA的D260/280值和产量,并对总RNA进行RT-PCR和凝胶电泳。结果显示,纯化的牛精子中不含体细胞,奶牛细管精液总RNA在2 h内完成提取,D260/280值为1.80,大于200 bp的总RNA产量为0.92μg/107个精子,精子总RNA进行RT-PCR,电泳后可得到清晰的SRY、LEP和RPS23基因3条带,不含18S rRNA基因条带。结果表明,离心层析柱法和Trizol一步法相结合可获得高质量的奶牛精子总RNA,且纯度和完整度高,可满足分子生物学研究的要求。

英文摘要：

Isolation of high-quality RNA is important for the study of sperm gene expression.Frozen straw semen from three bulls were purified by swimming up.The laminar analysis column method and Trizol single-step method were combined for RNA extraction from swimming-up bovine frozen-thaw sperm,and non-swimming up sperm as control.Sperm total RNA was analyzed with UV spectrophotometer,RT-PCR and garose gel electrophoresis.Results showed that the bovine sperm swimming-up from frozen straws were not contaminated by somatic cells,and high-quality total RNA was obtained in about 2 h.The value of D260/280 was 1.8,and the ≥200 bp total RNA extracted from bovine sperm yielded 0.92 μg/107sperm.Following RT-PCR,clear bands of SRY,LEP,and RPS23 of sperm cDNA were shown in a garose gel electrophoresis respectively,not a band of 18S rRNA.The purity and integrality of total RNA isolated from the bovine sperm of frozen straw with the method of combined the laminar analysis column with Trizol single-step were significantly satisfactory for the demands of molecular biological experiments.