Introduction Quantitative real-time PCR (qPCR) has been widely used for the quantification of target gene expression. One of the essential steps in qPCR assays is to select reference genes for normalizing the gene expression levels across the samples. In this post, we’ll use a small data set from the seawater trial of the AquaFly project. The candidate reference genes evaluated are beta-actin (actb), glyceraldehyde-3-phosphate dehydrogenase (gapdh), hypoxanthine phosphoribosyl transferase 1 (hprt1) and RNA polymerase II (rnapo2).