Abstract
Efficient RNA isolation is a prerequisite for gene expression studies and it has an increasingly important role in the study of plant–fungal pathogen interactions. However, RNA isolation is difficult in filamentous fungi. These organisms are notorious for their rigid cell walls and the presence of high levels of carbohydrates, excreted from the fungal cells during submerged growth, which interferes with the extraction procedures. Although many commercial kits are already available for RNA isolation, they do not provide, in most cases, enough amount of pure RNA to be used in upstream applications. In the present work, we propose an easy and efficient protocol for isolating total RNA from the filamentous fungus Mycosphaerella fijiensis, the most important foliar pathogen of Musa spp. varieties worldwide. In addition, we applied the proposed protocol to the isolation of total RNA from banana leaves infected with the pathogen. Our methodology was developed based on the SDS method with modifications including a carbohydrate precipitation step. The protocol resulted in high-quality total RNA, from fungal mycelium grown in PDB medium and infected banana leaves, suitable for further molecular studies. The proposed methodology is also applicable to the ascomycete fungus Passalora fulva (syn. Cladosporum fulvum).
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Acknowledgments
The authors thank José Gonzalez (Institute of Tropical Crops Research, Santo Domingo, Cuba) for preparation of fungal samples and we are grateful to Frank Maier (Department of Molecular Phytopathology and Genetics, Biocenter Klein Flottbek, Hamburg University, Germany) for providing us with the cytochrome b primers. This research was performed in the frame of the Institutional University Cooperation Programme with Universidad Central ¨Marta Abreu¨ de Las Villas, supported by the Flemish Interuniversity Council (IUC UCLV/VLIR).
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Aminael Sánchez-Rodríguez and Orelvis Portal contributed equally to the article.
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Sánchez-Rodríguez, A., Portal, O., Rojas, L.E. et al. An Efficient Method for the Extraction of High-Quality Fungal Total RNA to Study the Mycosphaerella fijiensis–Musa spp. Interaction. Mol Biotechnol 40, 299–305 (2008). https://doi.org/10.1007/s12033-008-9092-1
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DOI: https://doi.org/10.1007/s12033-008-9092-1