Identification of stable reference genes for quantitative PCR in koalas

Abstract

To better understand host and immune response to diseases, gene expression studies require identification of reference genes with stable expression for accurate normalisation. This study describes the selection and testing of reference genes with stable expression profiles in koala lymph node tissues across two genetically distinct koala populations. From the 25 most stable genes identified in transcriptome analysis, 11 genes were selected for verification using reverse transcription quantitative PCR, in addition to the commonly used ACTB and GAPDH genes. The expression data were analysed using stable genes statistical software - geNorm, BestKeeper, NormFinder, the comparative ΔCt method and RefFinder. All 13 genes showed relative stability in expression in koala lymph node tissues, however Tmem97 and Hmg20a were identified as the most stable genes across the two koala populations.

Selection of reference genes for quantitative real‐time PCR normalisation in largemouth bass Micropterus salmoides fed on alternative diets

“…The comprehensive ranking was given by RefFinder software and eef1a1 and tuba1 genes were recommended as the two most stable genes for different tissues. Recently, the website tool refFinder software has been used widely to assess suitable reference genes for various species (Auler et al, ; Kałużna et al, ; Sarker et al, ; Zhang et al, ) and human cell lines (Jacob et al, ; Kaszubowska et al, ) in many reports. By the analysis of four major algorithms, the results indicated that although an extent deviation existed in the tissue examples, eef1a1 and tuba1 could be considered the reliable reference genes for target gene expression in diet experiment.…”