Optimized methodology for obtention of high-yield and-quality RNA from the mycelium of the bioluminescent fungus Neonothopanus gardneri

Neonothopanus gardneri, also known as coconut flower mushroom (flor-de-coco), is a Brazilian bioluminescent basidiomycete found in Palm Forest, a transitional biome between the Amazonian Forest and Caatinga (Savanna like vegetation) in Northeast Brazil, especially in Piaui State. Recent advances toward the elucidation of fungal bioluminescence have contributed to the discovery of four genes (hisps, h3h, luz and cph) involved with the bioluminescence process, the so-called Caffeic Acid Cycle (CAC) and to develop biotechnological applications such autoluminescent tobacco plants and luciferase-based reporter genes. High-yield and-quality RNA-extraction methods are required for most of these purposes. Herein, four methods for RNA isolation from the mycelium of N. gardneri were evaluated: RNeasy (R) kit (QIAGEN), TRI+, TRI18G+, and TRI26G+. Highest RNA yield was observed for TRI18G+ and TRI26G+ methods, an increase of similar to 130% in comparison to the RNeasy (R) method and of similar to 40% to the TRI+ protocol. All the RNA samples showed good purity and integrity, except by gDNA contamination in RNA samples produced with the RNeasy (R) method. High quality of RNA samples was confirmed by successful cDNA synthesis and PCR amplification of the coding sequence of h3h gene, responsible for the hydroxylation of the precursor of fungal luciferin (3-hydroxyhispidin). Similarly, RT-qPCR amplification of ef-tu gene, related to the protein biosynthesis in the cell, was demonstrated from RNA samples. This is the first report of a reproducible, time-saving and low-cost optimized method for isolation of high-quality and-yield, DNA-free RNA from a bioluminescent fungus, but that can also be useful for other basidiomycetes.

Automated summary

This study evaluated a new RNA extraction method for obtaining high-quality and high-yield RNA from the bioluminescent fungus Neonothopanus gardneri. Compared to other methods, TRI18G+ and TRI26G+ methods yielded higher amounts of RNA, providing a reproducible, time-saving, and cost-effective solution.