人类精子高质量RNA提取方案的优化  

An optimizing protocol to isolate highly purified human sperm RNA

作　　者：于艳[1] 管群[1] 张斌[1] 闻姬[1] 杨丽霞[1] 菅福琴[1] 杨伟娜[1] 孙伟[1] 

机构地区：[1]山东中医药大学第二附属医院,济南市250001

出　　处：《实用医学杂志》2013年第21期3589-3593,共5页The Journal of Practical Medicine

基　　金：国家自然科学基金(编号:81102653);山东省自然科学基金(编号:ZR2012HL21;ZR2011HM013)

摘　　要：目的:人类精子所含RNA极少量,且由于精子中圆形细胞存在,使得分离精子RNA具有一定的挑战性。本研究旨在优化人类精子RNA的提取方案。方法:利用40%的单层梯度液及精子洗涤液纯化精子,使用TRIzol试剂和RNeasy Mini Kit提取RNA,反转录精子RNA,以cDNA为模板扩增PRM2、c-Kit、CD45、CDH1基因来确定所提精子RNA中是否存在基因组DNA或体细胞RNA的污染。结果:利用3倍体积的40%单层梯度液及精子洗涤液可以完全去除精液中体细胞的污染,利用TRIzol试剂提取RNA比RNeasy Mini Kit更有效。结论:人类精子中含有微量RNA,此方法既经济又有效,可以提取高质量的精子RNA,为人类男性生殖的功能基因组研究奠定基础。Objective The isolation of spermatozoal RNA is an challenge due to the small quantity of RNA in sperm and the soma cells in the ejaculate. The study was to find an optimizing protocol to isolate human sperm RNA. Methods Sperm were separated from somatic cells by centrifuging ejaculates with a single-layer 40% gradient system and sperm cleaning fluid. Sperm RNA were isolated with TRIzol reagent and RNeasy Mini Kit. Amplification of PRM2, c-Kit, CD45, CDH1 genes by RT-PCR was used to verify the contamination of genomic DNA or somatic cell RNA. Results Somatic cells in sperm could be completely eliminated by triple volume single-layer 40% gradient system and sperm cleaning fluid treatment. Sperm RNA isolation was more efficient with TRIzol reagent than with RNeasy Mini Kit. Conclusions Mieroamount of RNA is present in human sperm, and highly-quality RNA can be isolated by above protocol, which provided a foundation of functional genomic study on man fertility.

关 键 词：精子 RNA 男性不育 功能基因 

分 类 号：R440[医药卫生—诊断学]