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

Julie M. Old,Oselyne T.W. Ong,Lauren J. Young

doi:10.7717/peerj.2552

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; 18S rRNA, 18S; 28S rRNA, 28S; and ribosomal protein L13A, RPL13A) was investigated using SYBR Green and analysed with the geNorm application available in qBasePLUS software.ResultsGene 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.DiscussionBased 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.

Highlights

Fluorescence-based quantitative real-time polymerase chain reaction has the capacity to monitor the amplification of cDNA during thermocycling, starting with the use of ethidium bromide for the detection of fluorescence (Higuchi et al, 1992)
Five reference genes were amplified in four tissues, and all real-time PCR assays produced a single peak on the melting curve (GAPDH, Fig. S1; ACTB, Fig. S2; 18S, Fig. S3; 28S, Fig. S4 and ribosomal protein L13a (RPL13A), Fig. S5) and have been submitted into GenBank
We have successfully found stable reference genes in lung, small intestine and spleen tissue preparations from a dasyurid marsupial

Summary

Introduction

Fluorescence-based quantitative real-time polymerase chain reaction (qPCR) has the capacity to monitor the amplification of cDNA during thermocycling, starting with the use of ethidium bromide for the detection of fluorescence (Higuchi et al, 1992). 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. 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. 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

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