Selection and evaluation of reference genes for qRT-PCR in Inonotus obliquus

Real-time quantitative reverse transcription PCR (RT-qPCR) is one of the most effective tools for studying gene expression. <i>Inonotus obliquus</i> is a rare and edible medicinal fungus. Selecting an appropriate reference gene is crucial for researching its gene function. In this study, we selected 11 potential reference genes of <i>Inonotus obliquus</i>. Subsequently, we applied GeNorm, NormFinder, and BestKeeper algorithms to evaluate the expression stability of these 11 reference genes. Under varying carbon sources, <i>VPS</i> exhibited good stability and was suitable as an internal reference gene. Under different nitrogen sources, <i>RPB2</i> was the most stable reference gene. For varying growth factors, <i>PP2A</i> was the most stable reference gene. At different pH levels, <i>UBQ</i> showed the highest stability. Under different temperature conditions, <i>RPL4</i> was the most stable. In different strains, <i>RPL2</i> was the most stable reference gene, while <i>VAS</i> demonstrated the greatest stability across different growth stages. Overall, <i>VPS</i> was the most recommended reference gene for the entire sample set. This study provides a foundation for the further application of RT-qPCR in the gene expression analysis of <i>Inonotus obliquus</i>.