摘要
目的从扩增稳定性、扩增效率以及可靠性等多方面评估实时定量PCR（RtPCR）反应小体积的可行性,同时确定适合小体积体系的模板量。方法将实验分为10、15、20μL 3个体积组,每组分别采用0.1、0.2、0.3、0.4μL的大鼠cDNA为模板,进行大鼠-βactin mRNA的RtPCR扩增。通过扩增曲线和熔解曲线考察反应的稳定性,以梯度模板拟合标准曲线获得反应的扩增效率,其线性相关程度反映模板量是否合适。另外,构建大鼠降植烷诱导的关节炎（pristane-induced arthritis,PIA）模型,采用选定的反应体系,分别对模型组和正常组大鼠脾脏TNF-αmRNA进行扩增,通过TNF-α的相对表达趋势反映小体积扩增的可靠性。结果在大鼠-βactin mRNA的RtPCR扩增中,10、15、20μL 3个体积组均能特异稳定地进行扩增,并保证高扩增效率。而且每个体积组中的模板梯度表现出很好的线性相关趋势。同时选用10μL和20μL反应体积、0.2μL模板对关节炎大鼠脾脏进行TNF-αmRNA扩增,结果一致表现出了预期的显著性上调。结论实验证实10μL、15μL等小反应体积应用于RtPCR扩增中是可行的,具有较好的稳定性和可靠性,同时0.10.4μL cDNA模板在3种体系的合适模板范围内。这种较小的PCR反应体积,不仅节省了试剂和实验材料,对一些来之不易的临床标本尤为重要,也利于高通量大规模的RtPCR检测的实现。
Objective To evaluate the feasibility of real-time quantitative PCR（RtPCR） with small volume regarding the stability,efficiency and reliability of amplification,and determine the optimal quantity of cDNA template suitable for small PCR volume.Methods The experiment was carried out in 3 groups with 10,15 and 20μL reaction volume,respectively.In each group,rat β-actin mRNA was detected by RtPCR with 0.1,0.2,0.3 or 0.4μL rat cDNA as template,respectively.The amplification curve and melting curve were used to evaluate the reaction stability.The fitting of a curve of gradient templates against threshold cycle numbers was to show the reaction efficiency and the linear correlativity was to estimate the suitability of the template quantity.In addition,in order to estimate reliability,pristane-induced arthritis（PIA） rat model was established,and spleen TNF-α mRNA expression was detected by RtPCR with the selected reaction volumes.Results The amplification of rat β-actin mRNA was specific and stable in 10μL,15μL and 20μL PCR volume,and had a high efficiency.Furthermore,the standard curves fitted by 0.1-0.4μL gradient templates showed a significant linear correlation in each volume group.When the 10μL and 20μL PCR volumes,and 0.2μL cDNA templates were chosen,the TNF-α mRNA expression in PIA rat spleen showed significant upregulation in both two volume groups as anticipated.Conclusion The experiment shows that it is feasible in the RtPCR amplification to use the small reaction volume of 10μL and 15μL,which has good stability and reliability.And 0.1-0.4μL templates are all suitable for the reaction system.PCR with small volume can not only save the reagents and template,especially rare clinical specimens,but also is helpful for the realization of high-throughput reaction.
出处
《西安交通大学学报（医学版）》
CAS
CSCD
北大核心
2010年第2期177-180,共4页
Journal of Xi’an Jiaotong University（Medical Sciences）
基金
国家自然科学基金(No.30801027
30630058)
陕西省国际合作项目(2007-KW-06)~~
关键词
实时定量PCR
小反应体积
稳定性
可靠性
real-time quantitative PCR
small reactive volume
stability
reliability
