Goodrich Robert J, Anton Ester, Krawetz Stephen A
Department of Obstetrics and Gynecology, C. S. Mott Center for Human Growth and Development, Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI, USA.
Methods Mol Biol. 2013;927:385-96. doi: 10.1007/978-1-62703-038-0_33.
The isolation of spermatozoal RNA is a challenging procedure due to the intrinsic heterogeneous population of cells present in the ejaculate and the small quantity of RNA present in sperm. The transcriptome of these gametes includes a wide variety of messenger RNAs (mRNAs), small noncoding RNAs (sncRNAs), and highly fragmented ribosomal RNAs (rRNAs).The protocol described in this chapter to isolate both the mRNA and sncRNA fractions represents years of development towards automation. It combines a guanidinium thiocyanate-phenol-chloroform-based methodology to reduce the content of DNA and a column-based system. Both manual and semi-automated options are described, with preference given to automation for consistent results. A novel quality control procedure has been developed to assess the integrity and purity of the entire population of isolated mRNAs due to the absence of intact rRNAs.
由于精液中存在的细胞本质上具有异质性，且精子中存在的RNA量很少，因此分离精子RNA是一个具有挑战性的过程。这些配子的转录组包括各种各样的信使核糖核酸（mRNA）、小非编码核糖核酸（sncRNA）和高度碎片化的核糖体核糖核酸（rRNA）。本章中描述的分离mRNA和sncRNA组分的方案代表了多年来朝着自动化方向的发展。它结合了基于硫氰酸胍-苯酚-氯仿的方法来减少DNA含量和基于柱的系统。文中描述了手动和半自动两种选项，为了获得一致的结果，优先选择自动化。由于不存在完整的rRNA，已经开发了一种新的质量控制程序来评估分离出的全部mRNA的完整性和纯度。
