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A combined RNA preservation and extraction protocol for gene expression studies in cacao beans

Jocelyn De Wever (UGent) , Dieter Tulkens (UGent) , Jan Verwaeren (UGent) , Helena Everaert (UGent) , Hayley Rottiers (UGent) , Koen Dewettinck (UGent) , Steve Lefever (UGent) and Kathy Messens (UGent)
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Abstract
Despite the high economic importance of cacao beans, few RNA-based studies have been conducted on this plant material and hence no optimal RNA-extraction has been reported. Moreover, extraction of high-quality RNA from recalcitrant cacao bean tissue has shown many difficulties and requires optimization. Furthermore, cacao beans are mostly found at remote and under-resourced locations, which pressures the outsourcing of such analysis and thereby demands RNA-stable preservation and transportation of cacao beans. This study aims to select an appropriate RNA extraction and preservation/transportation method for cacao beans. For this purpose, three sample homogenization and five extraction protocols on cacao beans were compared. In addition, 13 preservation conditions-differing in tissue crushing degree, preservation method, duration, and temperature-were compared and evaluated. A comparative analysis revealed that CTAB-based homogenization and extraction outcompeted all tested commercial protocols in RNA yield and integrity, respectively. Preservation at -80 degrees C affected RNA quality the least, whereas freeze-drying was most suitable for transportation at room temperature for maximum 1 week. The cacao bean RNA obtained from the selected methods were compatible for downstream applications. The results of this study will facilitate on-field sampling and transportation of genetically sensitive cacao material prior to cacao bean transcriptomic studies. In addition, valuable insights on sample homogenization, extraction, preservation, and transportation have been provided, which is of interest to every plant geneticist.
Keywords
Plant Science, RNA, Theobroma cacaoL, extraction, freeze-drying, RNA quality numbers, preservation, transportation, cacao bean, HIGH-QUALITY RNA, THEOBROMA-CACAO, PLANT, IDENTIFICATION, TISSUE, INTEGRITY, CLONING, SEEDS, RICH

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MLA
De Wever, Jocelyn, et al. “A Combined RNA Preservation and Extraction Protocol for Gene Expression Studies in Cacao Beans.” FRONTIERS IN PLANT SCIENCE, vol. 11, 2020, doi:10.3389/fpls.2020.00992.
APA
De Wever, J., Tulkens, D., Verwaeren, J., Everaert, H., Rottiers, H., Dewettinck, K., … Messens, K. (2020). A combined RNA preservation and extraction protocol for gene expression studies in cacao beans. FRONTIERS IN PLANT SCIENCE, 11. https://doi.org/10.3389/fpls.2020.00992
Chicago author-date
De Wever, Jocelyn, Dieter Tulkens, Jan Verwaeren, Helena Everaert, Hayley Rottiers, Koen Dewettinck, Steve Lefever, and Kathy Messens. 2020. “A Combined RNA Preservation and Extraction Protocol for Gene Expression Studies in Cacao Beans.” FRONTIERS IN PLANT SCIENCE 11. https://doi.org/10.3389/fpls.2020.00992.
Chicago author-date (all authors)
De Wever, Jocelyn, Dieter Tulkens, Jan Verwaeren, Helena Everaert, Hayley Rottiers, Koen Dewettinck, Steve Lefever, and Kathy Messens. 2020. “A Combined RNA Preservation and Extraction Protocol for Gene Expression Studies in Cacao Beans.” FRONTIERS IN PLANT SCIENCE 11. doi:10.3389/fpls.2020.00992.
Vancouver
1.
De Wever J, Tulkens D, Verwaeren J, Everaert H, Rottiers H, Dewettinck K, et al. A combined RNA preservation and extraction protocol for gene expression studies in cacao beans. FRONTIERS IN PLANT SCIENCE. 2020;11.
IEEE
[1]
J. De Wever et al., “A combined RNA preservation and extraction protocol for gene expression studies in cacao beans,” FRONTIERS IN PLANT SCIENCE, vol. 11, 2020.
@article{8669171,
  abstract     = {{Despite the high economic importance of cacao beans, few RNA-based studies have been conducted on this plant material and hence no optimal RNA-extraction has been reported. Moreover, extraction of high-quality RNA from recalcitrant cacao bean tissue has shown many difficulties and requires optimization. Furthermore, cacao beans are mostly found at remote and under-resourced locations, which pressures the outsourcing of such analysis and thereby demands RNA-stable preservation and transportation of cacao beans. This study aims to select an appropriate RNA extraction and preservation/transportation method for cacao beans. For this purpose, three sample homogenization and five extraction protocols on cacao beans were compared. In addition, 13 preservation conditions-differing in tissue crushing degree, preservation method, duration, and temperature-were compared and evaluated. A comparative analysis revealed that CTAB-based homogenization and extraction outcompeted all tested commercial protocols in RNA yield and integrity, respectively. Preservation at -80 degrees C affected RNA quality the least, whereas freeze-drying was most suitable for transportation at room temperature for maximum 1 week. The cacao bean RNA obtained from the selected methods were compatible for downstream applications. The results of this study will facilitate on-field sampling and transportation of genetically sensitive cacao material prior to cacao bean transcriptomic studies. In addition, valuable insights on sample homogenization, extraction, preservation, and transportation have been provided, which is of interest to every plant geneticist.}},
  articleno    = {{992}},
  author       = {{De Wever, Jocelyn and Tulkens, Dieter and Verwaeren, Jan and Everaert, Helena and Rottiers, Hayley and Dewettinck, Koen and Lefever, Steve and Messens, Kathy}},
  issn         = {{1664-462X}},
  journal      = {{FRONTIERS IN PLANT SCIENCE}},
  keywords     = {{Plant Science,RNA,Theobroma cacaoL,extraction,freeze-drying,RNA quality numbers,preservation,transportation,cacao bean,HIGH-QUALITY RNA,THEOBROMA-CACAO,PLANT,IDENTIFICATION,TISSUE,INTEGRITY,CLONING,SEEDS,RICH}},
  language     = {{eng}},
  pages        = {{12}},
  title        = {{A combined RNA preservation and extraction protocol for gene expression studies in cacao beans}},
  url          = {{http://dx.doi.org/10.3389/fpls.2020.00992}},
  volume       = {{11}},
  year         = {{2020}},
}

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