CAJ | 학술논문

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、20μL 3个体积组,每组分别采用0.1、0.2、0.3、0.4μL的大鼠cDNA为模板,进行大鼠β-actin 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-α mRNA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β-actin mRNA的RtPCR扩增中,10、15、20μL 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10μL和20μL反应体积、0.2μL模板对关节炎大鼠脾脏进行TNF-α mRNA扩增,结果一致表现出了预期的显著性上调.结论实验证实10μL、15μL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0.1～0.4μL 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.
목적 종확증은정성、확증효솔이급가고성등다방면평고실시정량PCR(RtPCR)반응소체적적가행성,동시학정괄합소체적체계적모판량.방법 장실험분위10、15、20μL 3개체적조,매조분별채용0.1、0.2、0.3、0.4μL적대서cDNA위모판,진행대서β-actin mRNA적RtPCR확증.통과확증곡선화용해곡선고찰반응적은정성,이제도모판의합표준곡선획득반응적확증효솔,기선성상관정도반영모판량시부합괄.령외,구건대서강식완유도적관절염(pristane-induced arthritis, PIA)모형,채용선정적반응체계,분별대모형조화정상조대서비장TNF-α mRNA진행확증,통과TNF-α적상대표체추세반영소체적확증적가고성.결과 재대서β-actin mRNA적RtPCR확증중,10、15、20μL 3개체적조균능특이은정지진행확증,병보증고확증효솔.이차매개체적조중적모판제도표현출흔호적선성상관추세.동시선용10μL화20μL반응체적、0.2μL모판대관절염대서비장진행TNF-α mRNA확증,결과일치표현출료예기적현저성상조.결론 실험증실10μL、15μL등소반응체적응용우RtPCR확증중시가행적,구유교호적은정성화가고성,동시0.1～0.4μL cDNA모판재3충체계적합괄모판범위내.저충교소적PCR반응체적,불부절성료시제화실험재료,대일사래지불역적림상표본우위중요,야리우고통량대규모적RtPCR검측적실현.
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 20μL reaction volume, respectively. In each group, rat β-actin mRNA was detected by RtPCR with 0.1, 0.2, 0.3 or 0.4μL 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.