Comparison between droplet digital PCR and reverse transcription-quantitative PCR methods to measure ecotoxicology biomarkers. (May 2023)

• Record Type: Journal Article
• Authors: Baettig, Camille G.; Zirngibl, Martin; Smith, Kirsty F.; Lear, Gavin; Tremblay, Louis A.
• Is Part Of: Marine pollution bulletin. Volume 190(2023)
• Publication Date: 2023-05
• Subjects: Mussel -- Mytilus galloprovinciallis -- Copper -- Gene expression -- RT-qPCR -- ddPCR; Marine pollution -- Periodicals; Marine Biology -- Periodicals; Water Pollution -- Periodicals; Mer -- Pollution -- Périodiques; Publications périodiques; Pollution des mers; Lutte antipollution; Electronic journals; 363.7394
• DOI: 10.1016/j.marpolbul.2023.114829
• Languages: English
• ISSNs: 0025-326X

Abstract

Abstract: 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. Highlights: Step-by-step methodology to optimize and compare ddPCR and RT-qPCR is described. No statistical differences between the two methods was observed, suggesting good comparability. ddPCR is a suitable method to assess gene expression in an ecotoxicological context. RT-qPCR has a shorter processing time and remains more cost-effective.