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

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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.

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Acknowledgments

We thank Jenny Ruales of Escuela Politécnica Nacional, Quito (EPN), and Cocoa Research Institute of Ghana (CRIG) for making available their facilities, man power, and expertise on cacao bean selection and fermentation of the Ecuadorian and Ghanaian samples, respectively. Many thanks, especially to Kent Kobby Agyemang for his help in selecting Ghanaian cacao varieties. We thank the University of Ghana for allowing us access to their analytical equipment and the Centre for Plant Medicine Research for the freeze dryer. Many beloved thanks to my friends and colleagues, John Kongor and Michael Hinneh, for the personal guidance in Ghana and their availability. Besides, many thanks to Wietse Vancampenhout, for the optimization of RT-qPCR workflow and the validation of literature-based reference genes during his master thesis. Last but not least, we thank the CMGG staff, especially Kimberly Verniers, Thalia Van Laethem, and Dries Rombout, for the practical and technical assistance on the Fragment Analyzer, sequencing, and geNorm analysis, respectively.

Funding

This work was financially supported by a doctoral fellowship from the Special Research Fund (BOF16/DOC/338) and by Global minds at Ghent university (13V02318T). Mobility was funded by the Commission scientific research (CWO) (BOFFF22014000501) at the Faculty of Bioscience Engineering and the Research Foundation Flanders (FWO) (V405218N).

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Author notes

  1. Steve Lefever and Kathy Messens contributed equally to this work.

Authors and Affiliations

  1. Research Unit Molecular Biotechnology, Department of Biotechnology, Faculty of Bioscience Engineering, Ghent University, 9000, Ghent, Belgium

    Jocelyn De Wever, Tibo De Coninck, Helena Everaert, Hayley Rottiers & Kathy Messens

  2. Center for Medical Genetics Ghent (CMGG), Ghent University Hospital, 9000, Ghent, Belgium

    Jocelyn De Wever, Frauke Coppieters, Jo Vandesompele & Steve Lefever

  3. Laboratory of Food Technology and Engineering, Department of Food Technology, Safety and Health, Faculty of Bioscience Engineering, Ghent University, 9000, Ghent, Belgium

    Helena Everaert, Hayley Rottiers & Koen Dewettinck

  4. Ghana Technology University College, Accra, Ghana

    Emmanuel Ohene Afoakwa

  5. Cocoa Research Institute Ghana (CRIG), New-Tafo Akim, Ghana

    Stephen Yaw Opoku & Samuel Lowor

  6. Cancer Research Institute Ghent, Ghent University, 9000, Ghent, Belgium

    Jo Vandesompele & Steve Lefever

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Contributions

JDW and SLF created the experimental set-up. JDW wrote the manuscript together with TDC. JDW and SL conceived and designed the study as written in the main manuscript text. JDW and TDC equally contributed to the practical work conducted for this study. Both HE and HR offered their plant-associated expertise needed for this study, while SL and FC provided their expertise in the field of bioinformatics and next-generation sequencing, respectively. JV assisted with the geNorm software and provided a critical view on the statistical aspect of the paper. EOA, SYO, and SL have helped provide the cacao beans needed for this study, helped guide the fermentation trials and have provided access to their facilities, fermentation site, and analytical tools at CRIG and the University of Ghana, respectively. KM and KD supported and guided this study with their essential expertise, network, and resources in the field of cacao. All authors were involved in the revision of the draft manuscript and have agreed to the final content.

Corresponding author

Correspondence to Jocelyn De Wever.

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The authors declare that they have no conflict of interest.

Data archiving statement

All datasets, with exception of the raw RNA-sequencing data, analyzed within this study are included in the manuscript and the electronic supplementary material. The raw RNA-sequencing data is still under study and therefore not made available yet.

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Communicated by A. De La Torre

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De Wever, J., De Coninck, T., Everaert, H. et al. Selection and validation of reference genes for accurate RT-qPCR gene expression normalization in cacao beans during fermentation. Tree Genetics & Genomes 17, 7 (2021). https://doi.org/10.1007/s11295-021-01490-2

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  • DOI: https://doi.org/10.1007/s11295-021-01490-2

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