A modified culture medium and hyphae isolation method can increase quality of the RNA extracted from mycelia of a dimorphic fungal species

Abstract

The capability of RNA isolation with good efficiency and high quality is essential for a downstream application such as RNA sequencing. It requires successful cell culturing and an effective RNA isolation method. Although effective methods are available, production of the homogenous mycelia and extraction of good-quality mycelial RNA from true invasive hyphae, which penetrated into the agar plates, are difficult. To overcome these problems, the aim of this study was to develop technical modifications which allow production of homogenous mycelial biomass without extra stimuli agents and improve quality of the RNA extracted from the fungal hyphae. Our alternative culture medium was suitable for production both yeast-phase cells and hyphae of the Schizosaccharomyces japonicus and other dimorphic species, such as the Candida albicans, Saccharomyces cerevisiae, and Jaminaea angkorensis. To improve quality of the mycelial RNA, we developed an isolation procedure of the hyphal tip, which eliminated the unnecessary vacuoles-containing parts of the hyphae. To increase RNA quantity, we used glass beads in the RNA extraction protocol to achieve stronger breaking of the mycelial walls. All these modifications can also be useful for researchers working with other dimorphic fungi and can contribute to the higher comparability of the transcriptional data coming from yeast-phase cells and hyphae or even from different species.

Molecular and comparative genomic analyses reveal evolutionarily conserved and unique features of the Schizosaccharomyces japonicus mycelial growth and the underlying genomic changes

“…RNA was extracted from yeast-phase cells and hyphae. The culture conditions and modifications of the RNA isolation protocol (Lyne et al 2003 ) can be found in the previous article (Papp et al 2021 ). The most important modifications were the application of glass beads to achieve stronger break of the mycelial wall and RNA was extracted from cytoplasm-filled tips of the true invasive hyphae (instead of whole mycelia) which were grown on gelatin solidified culture medium (step-by-step protocol is in Papp et al 2021 ).…”

“…Total RNA was extracted from yeast-phase cells and hyphae as described previously (Papp et al 2021 ). cDNA synthesis was performed with the Thermo Scientific Revert Aid First Strand cDNA Synthesis Kit (K1621).…”

“…The Petri dishes were incubated at 30 °C. YPG (YEA solidified with 10% gelatin instead of agar) was used for the culturing of the yeast-phase and hyphalphase cells necessary for RNA isolation (Papp et al 2021).…”

How Does the Sample Preparation of Phytophthora infestans Mycelium Affect the Quality of Isolated RNA?

“…One of the reasons for the reduced integrity of isolated RNA could be the long process of collecting mycelium from the nutrient medium, which is associated with considerable damage. Therefore, using alternative cultivation techniques, such as those in [ 4 , 6 , 16 ], may help to improve the results.…”

“…Complications with RNA isolation are primarily due to the thick cell wall and active endogenous RNases, both issues having to be addressed at the earliest stages of the procedure, i.e., during cell grinding and lysis [ 1 ]. The success may depend on the isolation protocol [ 2 , 3 ], cultivation conditions [ 4 ], and even the material of the bead-beating system [ 1 ]. Most researchers favor grinding mycelium in liquid nitrogen [ 2 , 3 , 5 , 6 ], but this method is poorly scalable.…”