An accurate normalization strategy for RT-qPCR in Hypocrea jecorina (Trichoderma reesei).

Hypocrea jecorina is an important, filamentous fungus due to its effective production of hydrolytic enzymes. Gene expression studies provide deeper insight into environment sensing and cellular response mechanisms. Reverse transcription-quantitative PCR is a gene-specific and powerful tool to measure even minor changes in mRNA composition. An accurate normalization strategy is absolutely necessary for appropriate interpretation of reverse transcription-quantitative PCR results. One frequently applied strategy is the usage of a reference gene. Adequate reference genes for Hypocrea have not been published so far. By using the NormFinder and geNorm softwares, we evaluated the most stable genes amongst six potential reference genes in 34 samples from diverse cultivation conditions. Under those experimental conditions, sar1 encoding for a small GTPase was found to be the most stable gene, whereas act encoding for actin was not amongst the best validated ones. The influence of the reference system on the expression data is demonstrated by analysis of two target genes, encoding for the Xylanase regulator 1 and for Xylanase II. We further validated obtained xylanase 2 transcription rates with the corresponding enzyme activity.