Quantitative real-time polymerase chain reaction (qPCR) is probably the most used method for gene expression quantification because of its high sensitivity and specificity. Nevertheless, this technology can undergo experimental errors and variations. Normalization of the results using a reference gene is therefore necessary to minimize these variations. As the study of immune genes in bivalve mollusks has increased in the last years, the establishment of adequate and stable reference genes for bivalves is strongly required. We analyzed the behavior of four putative reference genes: ribosomal RNA 18S, actin, elongation factor 1 − α and α-tubulin. The suitability of these four genes as internal control for qPCR was evaluated in mussel (Mytilus galloprovincialis) and clam (Ruditapes philippinarum) hemocytes after bacterial challenge. Four independent approaches (BestKeeper, GeNorm, NormFinder and DeltaCt ) were used to assess the suitable genes for stable expression. For these particular circumstances, the most stable gene in hemocytes was elongation factor 1 − α for mussels and α-tubulin for clams.
