Ruiz-Villalba Adrián, van Pelt-Verkuil Elizabeth, Gunst Quinn D, Ruijter Jan M, van den Hoff Maurice Jb
Academic Medical Center, Department of Medical Biology, Meibergdreef 15, 1105AZ Amsterdam, The Netherlands.
Foundation of Applied Medical Research (FIMA), Area of Cell Therapy, University of Navarra, Avd Pío XII, 36 - 31008, Pamplona, Spain.
Biomol Detect Quantif. 2017 Nov 1;14:7-18. doi: 10.1016/j.bdq.2017.10.001. eCollection 2017 Dec.
Quantitative PCR allows the precise measurement of DNA concentrations and is generally considered to be straightforward and trouble free. However, a survey with 93 validated assays for genes in the Wnt-pathway showed that the amplification of nonspecific products occurs frequently and is unrelated to C or PCR efficiency values. Titration experiments showed that the occurrence of low and high melting temperature artifacts was shown to be determined by annealing temperature, primer concentration and cDNA input. To explore the range of input variations that occur in the normal use of the Cre assay these conditions were mimicked in a complete two-way design of template -plasmid DNA- and non-template -mouse cDNA- concentrations. These experiments showed that the frequency of the amplification of the correct product and the artifact, as well as the valid quantification of the correct product, depended on the concentration of the non-template cDNA. This finding questions the interpretation of dilution series in which template as well as non-template concentrations are simultaneously decreasing. Repetition of this cDNA concentration experiment with other templates revealed that exact reproduction qPCR experiments was affected by the time it takes to complete the pipetting of a qPCR plate. Long bench times were observed to lead to significantly more artifacts. However, the measurement of artifact-associated fluorescence can be avoided by inclusion of a small heating step after the elongation phase in the amplification protocol. Taken together, this trouble-shooting journey showed that reliability and reproducibility of qPCR experiments not only depends on standardization and reporting of the biochemistry and technical aspects but also on hitherto neglected factors as sample dilution and waiting times in the laboratory work flow.
定量聚合酶链反应（qPCR）可精确测量DNA浓度，通常被认为操作简单且没有问题。然而，一项针对Wnt信号通路中93个经过验证的基因检测方法的调查显示，非特异性产物的扩增经常发生，且与C值或PCR效率值无关。滴定实验表明，低熔点和高熔点假象的出现取决于退火温度、引物浓度和cDNA输入量。为了探究在Cre检测正常使用中出现的输入变化范围，在模板 - 质粒DNA - 和非模板 - 小鼠cDNA - 浓度的完全双向设计中模拟了这些条件。这些实验表明，正确产物和假象扩增的频率以及正确产物的有效定量取决于非模板cDNA的浓度。这一发现对稀释系列的解释提出了质疑，在稀释系列中模板和非模板浓度同时降低。用其他模板重复这个cDNA浓度实验发现，qPCR实验的精确重现受到完成qPCR板移液所需时间的影响。观察到较长的实验台操作时间会导致产生明显更多的假象。然而，通过在扩增方案的延伸阶段后加入一个小的加热步骤，可以避免与假象相关的荧光测量。综上所述，这段故障排除历程表明，qPCR实验的可靠性和可重复性不仅取决于生物化学和技术方面的标准化及报告，还取决于实验室工作流程中样本稀释和等待时间等迄今被忽视的因素。
