奶牛圆形、长形、成熟精子细胞微量RNA制备和含量分析

doi:doi: 10.11843/j.issn.0366-6964.2016.08.004

摘要/Abstract

摘要：

本研究旨在探究奶牛变形精子细胞获取方法、建立稳定奶牛精子RNA提取体系及分析单个生精细胞RNA含量变化。采用激光显微捕获技术结合冰冻切片，采集奶牛圆形、长形精子细胞样品，利用附睾尾精子浮游法获得成熟精子，通过对已报道的小鼠、奶牛及人类精子RNA制备方法的尝试和比较，获得RNeasy Kit最佳奶牛精子RNA制备方法，并用PicoPure RNA Isolation Kit对圆形和长形精子细胞进行RNA制备，筛选去除白细胞、上皮细胞等体细胞污染及基因组DNA污染的反转录聚合酶链式反应（RT-PCR）检测方法，并对生精细胞RNA含量进行单因素方差分析。结果表明，激光显微切割技术可有效捕获圆形、长形精子细胞；通过RNeasy Kit获得的牛精子RNA纯度高、质量好，单个精子RNA含量为0.023~0.025 pg；同时，随精子细胞变形，其RNA含量显著降低，其中圆形精子到成熟精子RNA含量降低75倍。本研究从复杂样品取样、微量RNA细胞转录本制备与检测等方面，为精子发生与变形机理研究提供重要参考。

关键词: 生精细胞, 转录本, 激光显微切割, 附睾, 表达

Abstract:

To explore the method to capture dairy cattles round and elongated spermatids and build the stable system extracting sperm RNA and analyzing the RNA quantity of spermatogenic cells，the round and elongated spermatids were captured by laser capture microdissection (LCM) combined with the technology of frozen section，and sperm samples were obtained using the method of sperm floating from epididymids.Compared the reported sperm RNA preparation methods，a better RNA preparation result was obtained from dairy cattle sperm using RNeasy Kit，and the better RNA preparation results were also obtained from round and elongated spermatids using PicoPure RNA Isolation Kit.Besides，the removal results of contaminated somatic cells and genomic DNA were tested by reverse transcription polymerase chain reaction（RT-PCR）.The one-way analysis of variance showed that the round and elongated spermatids were successfully captured by LCM，the high quality RNA was extracted from sperm using RNeasy Kit，and the RNA quantity of each sperm was 0.023-0.025 pg.The RNA quantity decreased by 750-fold from round to elongated spermatids.The exploration that capture spermatogenic cells from complex tissues and extract RNA from less RNA containing cells will provide an important reference for the study on mechanism of spermatogenesis and spermiogenesis.

Key words: spermatogenic cells, transcripts, laser capture microdissection, epididymis, expression

中图分类号:

S814

任小霞，陈晓丽，李秀岭，王艳，秦彤，王栋. 奶牛圆形、长形、成熟精子细胞微量RNA制备和含量分析[J]. 畜牧兽医学报, 2016, 47(8): 1546-1554.

REN Xiao-xia，CHEN Xiao-li，LI Xiu-ling，WANG Yan，QIN Tong，WANG Dong. The Preparation and Quantity Analysis of Trace RNA from Round Spermatids，Elongated Spermatids and Mature Sperm of Dairy Cattle[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(8): 1546-1554.

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