Baettig Camille G, Zirngibl Martin, Smith Kirsty F, Lear Gavin, Tremblay Louis A
School of Biological Sciences, University of Auckland, Auckland, New Zealand; Cawthron Institute, Nelson, New Zealand.
Cawthron Institute, Nelson, New Zealand.
Mar Pollut Bull. 2023 May;190:114829. doi: 10.1016/j.marpolbul.2023.114829. Epub 2023 Mar 21.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is currently the gold-standard technique for detecting and quantifying messenger RNA. However, without proper validation, the method may produce artefactual and non-reproducible cycle threshold values generating poor-quality data. The newer droplet digital PCR (ddPCR) method allows for the absolute quantification of targeted nucleic acids providing more sensitive and accurate measurements without requiring external standards. This study compared these two PCR-based methods to measure the expression of well-documented genes used in ecotoxicology studies. We exposed Mediterranean mussels (Mytilus galloprovincialis) to copper and analyzed gene expression in gills and digestive glands using RT-qPCR and ddPCR assays. A step-by-step methodology to optimize and compare the two technologies is described. After ten-fold serial complementary DNA dilution, both RT-qPCR and ddPCR exhibited comparable linearity and efficiency and produced statistically similar results. We conclude that ddPCR is a suitable method to assess gene expression in an ecotoxicological context. However, RT-qPCR has a shorter processing time and remains more cost-effective.
逆转录定量聚合酶链反应（RT-qPCR）是目前检测和定量信使核糖核酸的金标准技术。然而，如果没有适当验证，该方法可能会产生人为的、不可重复的循环阈值，从而生成质量较差的数据。更新的液滴数字PCR（ddPCR）方法可对靶向核酸进行绝对定量，无需外部标准即可提供更灵敏、准确的测量。本研究比较了这两种基于PCR的方法，以测量生态毒理学研究中常用的、记录充分的基因的表达。我们将地中海贻贝（Mytilus galloprovincialis）暴露于铜中，并使用RT-qPCR和ddPCR分析鳃和消化腺中的基因表达。文中描述了优化和比较这两种技术的逐步方法。在进行十倍系列互补DNA稀释后，RT-qPCR和ddPCR均表现出相当的线性和效率，并产生了统计学上相似的结果。我们得出结论，ddPCR是在生态毒理学背景下评估基因表达的合适方法。然而，RT-qPCR的处理时间更短，且成本效益更高。
