Selection and validation of reference genes for accurate RT-qPCR gene expression normalization in cacao beans during fermentation

Abstract

To date, little attention has been paid to the genotypic plasticity and influence of the fermentation process on gene functions and biological processes in cacao beans. The primary tools for such analyses are gene expression studies with reverse transcription quantitative PCR (RT-qPCR). While this is a well-appreciated technique, it is only reliable when considering the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, which is unfortunately barely applied in plant sciences and non-existent in cacao-related studies. In this study, an appropriate from bean to RT-qPCR protocol was developed. In total, sixty-five candidate reference gene (RG) assays were validated. These assays were either adopted from literature (traditional "housekeeping" genes) or based on RNA-sequencing data (novel). After validation, three novel reference genes (SUGP1, NAP1, SGT1) were recommended for normalization of gene expression within fermented cacao beans. The suitability of the novel candidates surpassed the traditional housekeeping genes. In addition, these assays seemed largely unaffected by RNA integrity. This is the first study to establish a standardized RT-qPCR workflow on cacao beans during fermentation, facilitating future studies. We recommend similar MIQE-based approaches for future gene expression studies on other organisms for miscellaneous objectives.