摘要
目的:建立并优化SYBR GreenI实时RT-PCR体系,定量检测人、小鼠成熟精子中的CatSper1 mRNA。方法:用TRIzol分别提取人、小鼠成熟精子中的总RNA,逆转录后用SYBR GreenI实时PCR定量检测CatSper1 mRNA。SYBR GreenI实时PCR采用普通PCR试剂,加入SYBR GreenI染料,优化退火温度、Mg2+浓度及上、下游引物比例,并在PCR循环时采用四步法以消除引物二聚体的影响。优化完成后用不同浓度的精子cDNA为模板做标准曲线,以检测SYBR GreenI实时PCR的扩增效率。结果:定量检测CatSper1 mRNA的SYBR GreenI实时PCR体系适宜退火温度、Mg2+浓度及上、下游引物比例分别为63℃、3.0mmol/L和1∶1,四步法中采集荧光的温度为88℃。优化后用人和小鼠精子cDNA为模板做标准曲线分别为Y=-3.402log(X)+25.99和Y=-3.409log(X)+24.09,扩增效率分别为96.8%和96.5%,可定量检测人、小鼠成熟精子中的CatSper1 mRNA。结论:用普通的逆转录及PCR系统和试剂,建立了一种方便、廉价、可靠的SYBR GreenI实时荧光定量RT-PCR系统,可用于人、小鼠精子中CatSper1 mRNA定量检测。
Objective： To establish and optimize a real time RT-PCR system for determining the transcript levels of CatSperl in human and mouse mature spermatozoa containing microamount of RNA. Methods ： 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 CatSperl 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, Mg^2＋ 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 CatSperl mRNA levels in the human and mouse mature spermatozoa. Results ： The optimal conditions for real time RT-PCR, that is, annealing temperature, Mg^2 ＋ concentration and the ratio between forward and reverse primers were 63 ℃, 3.0 mmol/L and 1：1 respectively. The fluorescence measurement temperature was 88 ℃. The standard curves were Y = - 3. 402log（X） ＋ 25.99 and Y = -3. 409log（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 CatSperl mRNA levels in the human and mouse mature spermatozoa could be determined ac- cording to the standard curve. Conclusion： 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.
出处
《中华男科学杂志》
CAS
CSCD
2007年第11期969-974,共6页
National Journal of Andrology
基金
国家"十五"科技攻关项目(2004BA720A33-01)
湖北省卫生厅科研基金项目(JX2B03)
