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Background  Research using the zebrafish model has experienced a rapid growth in recent years. Although real-time reverse transcription PCR (QPCR), normalized to an internal reference ("housekeeping") gene, is a frequently used method for quantifying gene expression changes in zebrafish, many commonly used housekeeping genes are known to vary with experimental conditions. To identify housekeeping genes that are stably expressed under different experimental conditions, and thus suitable as normalizers for QPCR in zebrafish, the present study evaluated the expression of eight commonly used housekeeping genes as a function of stage and hormone/toxicant exposure during development, and by tissue type and sex in adult fish.    Results  QPCR analysis was used to quantify mRNA levels of  bactin1, tubulin alpha 1(tuba1), glyceraldehyde-3-phosphate dehydrogenase (gapdh), glucose-6-phosphate dehydrogenase (g6pd), TATA-box binding protein (tbp), beta-2-microglobulin (b2m), elongation factor 1 alpha (elfa) , and  18s ribosomal RNA (18s)  during development (2 – 120 hr postfertilization, hpf); in different tissue types (brain, eye, liver, heart, muscle, gonads) of adult males and females; and after treatment of embryos/larvae (24 – 96 hpf) with commonly used vehicles for administration and agents that represent known environmental endocrine disruptors. All genes were found to have some degree of variability under the conditions tested here. Rank ordering of expression stability using geNorm analysis identified  18s ,  b2m , and  elfa  as most stable during development and across tissue types, while  gapdh, tuba1 , and  tpb  were the most variable. Following chemical treatment,  tuba1, bactin1 , and  elfa  were the most stably expressed whereas  tbp, 18s , and  b2m  were the least stable. Data also revealed sex differences that are gene- and tissue-specific, and treatment effects that are gene-, vehicle- and ligand-specific. When the accuracy of QPCR analysis was tested using different reference genes to measure suppression of  cyp19a1b  by an estrogen receptor antagonist and induction of  cyp1a  by an arylhydrocarbon receptor agonist, the direction and magnitude of effects with stable and unstable genes differed.    Conclusion  This study provides data that can be expected to aid zebrafish researchers in their initial choice of housekeeping genes for future studies, but underlines the importance of further validating housekeeping genes for each new experimental paradigm and fish species.

Characterization of housekeeping genes in zebrafish: male-female differences and effects of tissue type, developmental stage and chemical treatment