Li Hong-gang, Ding Xiao-fang, Xiong Cheng-liang
Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430030 People's Republic of China.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2008 Aug;25(4):465-8.
To study the effect of the split of sperm nuclei on the yield of RNA from human sperm.
Human sperm were purified by two sequential centrifugations through 40:80 discontinuous gradients of Percoll. Human leukocytes separated from peripheral blood were used as the control. Total RNAs from purified sperm and leukocytes were extracted with both TRIzol and RNeasy Kit. The RNAs from equal number of cells were reverse-transcribed, and quantified by the levels of beta-ACTIN mRNA, which were evaluated by real time polymerase chain reaction.
TRIzol failed to digest majority of sperm nuclei even the incubation time was prolonged to 1 h, while no sperm nucleus was found under the light microscope after 1 min digestion with RLT buffer of the RNeasy Kit. Both reagents can digest the nuclei of human leukocytes well. The amount of RNA per 10(6) sperms isolated with RNeasy Kit (149.8+/-24.5 ng) was 4-fold higher (P=0.01) than that extracted with TRIzol (35.5+/-4.0 ng per 10(6) spermatozoa; n=3). The similar yields of the leukocyte RNAs extracted with RNeasy Kit and TRIzol [(765.5+/-229.8) and (958.8+/-201.0) ng per 10(6) cells respectively; n=3, P=0.168] excluded the possibility of different efficacy of these two reagents in RNA isolation.
The split of sperm nuclei is important to the yield of RNA in the human sperm RNA extraction. The nucleus may be the major area for human sperm RNA repositories.
研究精子细胞核裂解对人精子RNA产量的影响。
通过在Percoll 40:80不连续梯度中进行两次连续离心纯化人精子。将从外周血中分离出的人白细胞用作对照。用TRIzol和RNeasy试剂盒从纯化的精子和白细胞中提取总RNA。将等量细胞的RNA进行逆转录，并通过β-肌动蛋白mRNA水平进行定量，通过实时聚合酶链反应进行评估。
即使孵育时间延长至1小时，TRIzol也未能消化大多数精子细胞核，而用RNeasy试剂盒的RLT缓冲液消化1分钟后，在光学显微镜下未发现精子细胞核。两种试剂都能很好地消化人白细胞的细胞核。用RNeasy试剂盒分离的每10⁶个精子的RNA量（149.8±24.5 ng）比用TRIzol提取的量高4倍（P = 0.01）（每10⁶个精子35.5±4.0 ng；n = 3）。用RNeasy试剂盒和TRIzol提取的白细胞RNA产量相似[分别为每10⁶个细胞（765.5±229.8）和（958.8±201.0）ng；n = 3，P = 0.168]，排除了这两种试剂在RNA分离中不同功效的可能性。
精子细胞核裂解对人精子RNA提取中的RNA产量很重要。细胞核可能是人精子RNA储存的主要区域。
