An efficient method for isolation of high-quality RNA from mycelium of toxigenic fungi Fusarium sp.

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Abstract

Here, we describe a rapid and relatively cost-efficient method for the isolation and purification of RNA from mycelium of two species of plant-pathogenic fungi of Fusarium genus with different morphological and biochemical properties, F. graminearum and F. coffeatum. The method involves the use of guanidine hydrochloride-based buffer and spin columns from a commercial plasmid DNA extraction kit, and can be applied to both mycelia grown on nutrient agar media and liquid cultures of fungi. The yield of RNA isolated using the proposed protocol was 4–14 µg/100 mg of mycelium dry weight with RIN values up to 8.4. When optimizing the method, we propose to carry out a pre-lyophilization procedure, as well as the use of an RNAse inhibitor when isolating from cultures at late growth stages.

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About the authors

А. А. Stakheev

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Author for correspondence.
Email: stakheev.aa@gmail.com
Russian Federation, Moscow

D. Yu. Ryazantsev

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: stakheev.aa@gmail.com
Russian Federation, Moscow

N. G. Gabrielyan

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: stakheev.aa@gmail.com
Russian Federation, Moscow

A. V. Poluboyarinova

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: stakheev.aa@gmail.com
Russian Federation, Moscow

М. E. Taliansky

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: stakheev.aa@gmail.com
Russian Federation, Moscow

S. K. Zavriev

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: stakheev.aa@gmail.com
Russian Federation, Moscow

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Supplementary files

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2. Fig. 1. Results of the analysis of the integrity of RNA of F. graminearum (a) and F. coffeatum (b), performed on an Agilent 2100 Bioanalyzer (Agilent Technologies, USA). RNA was isolated from fungal mycelia on the 4th day of growth in a liquid nutrient medium.

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3. Fig. 2. Electropherogram of RNA samples isolated from fungal mycelia on the 7th day of growth in liquid nutrient media. To the left of the marker (M) are samples isolated without the RiboCare RNase inhibitor, to the right – with the addition of the inhibitor. 1, 5 – F. graminearum, RNeasy Plant Mini Kit; 2, 6 – F. graminearum, method using 8 M guanidine hydrochloride; 3, 7 – F. coffeatum, RNeasy Plant Mini Kit; 4, 8 – F. coffeatum, method using 8 M guanidine hydrochloride.

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4. Fig. 3. Graphs of the dependence of PCR threshold cycles on the degree of dilution of F. graminearum (a) and F. coffeatum (b) cDNA. The SD value is indicated next to each point.

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