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

Abstract

BackgroundReference 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.MethodsThe 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;18SrRNA,18S; 28SrRNA, 28S;and ribosomal protein L13A,RPL13A) was investigated using SYBR Green and analysed with the geNorm application available in qBasePLUSsoftware.ResultsGene stability for juvenile and adult tissue samples combined show thatGAPDHwas most stable in liver and lung tissue, and18Sin small intestine and spleen. While all reference genes were suitable for small intestine and spleen tissues, all reference genes except28Swere stable for lung and only18Sand28Swere stable for liver tissue. Separating the two age groups, we found that two different reference genes were considered stable in juveniles (ACTBandGAPDH) and adults (18Sand28S), andRPL13Awas not stable for juvenile small intestine tissue. Except for28S, all reference genes were stable in juvenile and adult lungs, and all five reference genes were stable in spleen tissue.DiscussionBased on expression stability,ACTBandGAPDHare 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 butRPL13Awas not suitable in juveniles.