Validation of hisH4 and cox5 reference genes for RT-qPCR analysis of gene expression in Aspergillus flavus under aflatoxin conducive and non-conducive conditions.

Aspergillus flavus is an environmental pathogen that produces highly carcinogenic aflatoxins. Biosynthesis of aflatoxins is affected by external factors such as pH, temperature, carbon source and nitrogen source. Real-Time PCR (RT-qPCR) is a powerful technique used to detect minute changes in gene expression of a target gene in comparison to one or more reference genes. Several candidate genes were analysed to determine their suitability for use as reference genes for analysing gene expression in A. flavus via RT-qPCR under various aflatoxin conducive and non-conducive conditions. BestKeeper analysis indicated that histone H4 (hisH4) and cytochrome C oxidase subunit V (cox5) were suitable reference genes for analysis of gene expression in A. flavus via RT-qPCR. This was further confirmed by REST2009 analysis of hisH4 and cox5 stability. Furthermore, REST2009 was used to predict which gene or gene combination would be the best reference gene/s for RT-qPCR expression analysis under each treatment condition tested in this study.