Van Heetvelde Mattias, Van Loocke Wouter, Trypsteen Wim, Baert Annelot, Vanderheyden Katrien, Crombez Brecht, Vandesompele Jo, De Leeneer Kim, Claes Kathleen B M
Center for Medical Genetics Ghent, Ghent University Hospital, Ghent, Belgium.
Department of Basic Medical Sciences, Ghent University, Ghent, Belgium.
Biomol Detect Quantif. 2017 Sep 20;13:40-48. doi: 10.1016/j.bdq.2017.09.001. eCollection 2017 Sep.
For the relative quantification of isoform expression, RT-qPCR has been the gold standard for over a decade. More recently, digital PCR is becoming widely implemented, as it is promised to be more accurate, sensitive and less affected by inhibitors, without the need for standard curves. In this study we evaluated RT-qPCR versus RT-droplet digital PCR (ddPCR) for the relative quantification of isoforms in controls and carriers of the splice site mutation c.212+3A>G, associated with increased expression of several isoforms.
RNA was extracted from EBV cell lines of controls and heterozygous c.212+3A>G carriers. Transcript-specific plasmids were available to determine the efficiency, precision, reproducibility and accuracy of each method.
Both ddPCR and RT-qPCR were able to accurately quantify all targets and showed the same LOB, LOD and LOQ; also precision and reproducibility were similar. Both techniques have the same dynamic range and linearity at biologically relevant template concentrations. However, a significantly higher cost and workload was required for ddPCR experiments.
Our study recognizes the potential and validity of digital PCR but shows the value of a highly optimized qPCR for the relative quantification of isoforms. Cost efficiency and simplicity turned out to be better for RT-qPCR.
在过去十多年里，逆转录定量聚合酶链反应（RT-qPCR）一直是异构体表达相对定量的金标准。最近，数字PCR正得到广泛应用，因为它有望更准确、灵敏，且受抑制剂影响较小，无需标准曲线。在本研究中，我们评估了RT-qPCR与逆转录液滴数字PCR（RT-ddPCR）用于对照以及与几种异构体表达增加相关的剪接位点突变c.212+3A>G携带者中异构体的相对定量。
从对照和杂合c.212+3A>G携带者的EBV细胞系中提取RNA。有转录本特异性质粒可用于确定每种方法的效率、精密度、重现性和准确性。
ddPCR和RT-qPCR都能够准确地定量所有靶标，并且显示出相同的检测限（LOB）、检测下限（LOD）和定量下限（LOQ）；精密度和重现性也相似。在生物学相关模板浓度下，两种技术具有相同的动态范围和线性。然而，ddPCR实验需要显著更高的成本和工作量。
我们的研究认可数字PCR的潜力和有效性，但显示了高度优化的qPCR用于异构体相对定量的价值。结果表明，RT-qPCR在成本效益和简便性方面更具优势。
