Comparison between droplet digital PCR and reverse transcription-quantitative PCR methods to measure ecotoxicology biomarkers

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 tech-nologies 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.

Automated summary

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the current gold-standard technique for detecting and quantifying messenger RNA. Droplet digital PCR (ddPCR) allows for more sensitive and accurate measurements of targeted nucleic acids without external standards. This study compared these two PCR-based methods and concluded that ddPCR is suitable for assessing gene expression in an ecotoxicological context, while RT-qPCR is more time-efficient and cost-effective.