Validation of Reference Genes for RT-qPCR in Marine Bivalve Ecotoxicology: Systematic Review and Case Study

Reverse transcription real-time quantitative PCR (RT-qPCR) is the predominant method of choice for the quantification of mRNA transcripts of a selected gene of interest. Here reference genes are commonly used to normalize non-biological variation in mRNA levels and their appropriate selection is therefore essential for the accurate interpretation the collected data. In recent years the use of multiple validated references genes has been shown to substantially increase the robustness of the normalization. It is therefore considered good practice to experimentally validate putative reference genes under specific experimental conditions, determine the optimal number of reference genes to be emp