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Background  Reference genes serve an important role as an endogenous control/standard for data normalisation in gene expression studies. Although reference genes have recently been suggested for marsupials, independent analysis of reference genes on different immune tissues is yet to be tested. Therefore, an assessment of reference genes is needed for the selection of stable, expressed genes across different marsupial tissues.    Methods  The study was conducted on red-tailed phascogales ( Phascogale calura ) using five juvenile and five adult males. The stability of five reference genes (glyceraldehyde-3-phosphate dehydrogenase,  GAPDH ;  β -actin,  ACTB ;  18S  rRNA,  18S; 28S  rRNA , 28S;  and ribosomal protein L13A,  RPL13A ) was investigated using SYBR Green and analysed with the geNorm application available in qBase PLUS  software.    Results  Gene stability for juvenile and adult tissue samples combined show that  GAPDH  was most stable in liver and lung tissue, and  18S  in small intestine and spleen. While all reference genes were suitable for small intestine and spleen tissues, all reference genes except  28S  were stable for lung and only  18S  and  28S  were stable for liver tissue. Separating the two age groups, we found that two different reference genes were considered stable in juveniles ( ACTB  and  GAPDH ) and adults ( 18S  and  28S ), and  RPL13A  was not stable for juvenile small intestine tissue. Except for  28S , all reference genes were stable in juvenile and adult lungs, and all five reference genes were stable in spleen tissue.    Discussion  Based on expression stability,  ACTB  and  GAPDH  are suitable for all tissues when studying the expression of marsupials in two age groups, except for adult liver tissues. The expression stability between juvenile and adult liver tissue was most unstable, as the stable reference genes for juveniles and adults were different. Juvenile and adult lung, small intestine and spleen share similar stable reference genes, except for small intestine tissues where all reference genes were stable in adults but  RPL13A  was not suitable in juveniles.

Evaluation of reference genes for gene expression in red-tailed phascogale (Phascogale calura) liver, lung, small intestine and spleen