Li Hong-gang, Liao Ai-hua, Kong Xiang-bin, Hu Lian, Xiong Cheng-liang
Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, Wu-han, Hubei 430030, China.
Zhonghua Nan Ke Xue. 2007 Nov;13(11):969-74.
To establish and optimize a real time RT-PCR system for determining the transcript levels of CatSper1 in human and mouse mature spermatozoa containing microamount of RNA.
Total RNA of human and mouse mature spermatozoa was isolated by using TRIzol reagent and reversely transcribed to complementary DNA respectively. Primers for real time RT-PCR were designed in the homologous area of the human and mouse CatSper1 mRNAs. Human sperm complementary DNA was used as the template to the optimize the conditions for SYBR Green I real time RT-PCR, including annealing temperature, Mg2+ concentration, fluorescence measurement temperature and the ratio between forward and reverse primers. The standard curve was constructed with serial dilutions of complementary DNA from human sperm to ascertain the amplification efficiency of SYBR Green I real time PCR and to quantitate the CatSper1 mRNA levels in the human and mouse mature spermatozoa.
The optimal conditions for real time RT-PCR, that is, annealing temperature, Mg2+ concentration and the ratio between forward and reverse primers were 63 degrees C, 3.0 mmol/L and 1:1 respectively. The fluorescence measurement temperature was 88 degrees C. The standard curves were Y = -3.402 log (X) + 25.99 and Y = -3.409 log(X) + 24.09 in the human sperm cDNA and mouse sperm cDNA as the template, with amplification efficiency of 96.8% and 96.5% respectively. The R2 value (an indicator of the quality of the fit of the standard curve to the standard data points plotted) of both standard curves was 0.998. The CatSper1 mRNA levels in the human and mouse mature spermatozoa could be determined according to the standard curve.
The general RT-PCR system, by adding SYBR Green I and optimizing its conditions, could be used to quantitate the mRNA levels in both human and mouse mature spermatozoa.
建立并优化一种实时逆转录聚合酶链反应（RT-PCR）系统，用于测定人和小鼠成熟精子中微量RNA的CatSper1转录水平。
使用TRIzol试剂分别提取人和小鼠成熟精子的总RNA，并分别逆转录为互补DNA。在人和小鼠CatSper1 mRNA的同源区域设计实时RT-PCR引物。以人精子互补DNA为模板，优化SYBR Green I实时RT-PCR条件，包括退火温度、Mg2+浓度、荧光测量温度以及上下游引物比例。用人精子互补DNA的系列稀释液构建标准曲线，以确定SYBR Green I实时PCR的扩增效率，并定量人和小鼠成熟精子中的CatSper1 mRNA水平。
实时RT-PCR的最佳条件，即退火温度、Mg2+浓度和上下游引物比例分别为63℃、3.0 mmol/L和1:1。荧光测量温度为88℃。以人精子cDNA和小鼠精子cDNA为模板的标准曲线分别为Y = -3.402 log (X) + 25.99和Y = -3.409 log(X) + 24.09，扩增效率分别为96.8%和96.5%。两条标准曲线的R2值（标准曲线与绘制的标准数据点拟合质量的指标）均为0.998。可根据标准曲线测定人和小鼠成熟精子中的CatSper1 mRNA水平。
通过添加SYBR Green I并优化其条件的通用RT-PCR系统，可用于定量人和小鼠成熟精子中的mRNA水平。
