Efficient Extraction Method for High Quality Fungal RNA from Complex Lignocellulosic Substrates

  • Miia R. Mäkelä
  • Kristiina Hildén
Part of the Methods in Molecular Biology book series (MIMB, volume 1775)


Here we describe an efficient and reproducible method for the extraction of fungal RNA from complex lignocellulose containing materials. The fungal cells are snap-frozen and disrupted in chaotropic guanidinium thiocyanate buffer, after which the extracted RNA is isolated by using CsCl gradient ultracentrifugation. By lowering the pH of the extraction buffer, the procedure is also suitable for sample materials rich in humic acids. The method results in high quantity and quality RNA that is separated from endogenous contaminants (e.g., RNases) and substances derived from plant biomass (e.g., colored aromatic compounds). In addition, no further steps such as DNase treatment are needed. The extracted RNA is highly suitable for downstream gene expression analyses such as RNA sequencing.


Lignocellulose Plant biomass Fungi Basidiomycetes Cesium chloride RNA extraction Ultracentrifugation 


  1. 1.
    England LS, Trevors JT (2003) The microbial DNA cycle in soil. Riv Biol 96:317–326PubMedGoogle Scholar
  2. 2.
    England LS, Trevors JT, Holmes SB (2001) Extraction and detection of baculoviral DNA from lake water, detritus and forest litter. J Appl Microbiol 90:630–636CrossRefPubMedGoogle Scholar
  3. 3.
    Sayler GS, Fleming JT, Nivens DE (2001) Gene expression monitoring in soils by mRNA analysis and gene lux fusions. Curr Opin Biotechnol 12:455–460CrossRefPubMedGoogle Scholar
  4. 4.
    Trevors JT (1996) Nucleic acids in the environment. Curr Opin Biotechnol 7:331–336CrossRefPubMedGoogle Scholar
  5. 5.
    Leite GM, Magan N, Medina Á (2012) Comparison of different bead-beating RNA extraction strategies: an optimized method for filamentous fungi. J Microbiol Methods 88:413–418CrossRefPubMedGoogle Scholar
  6. 6.
    Mettel C, Kim Y, Shrestha PM et al (2010) Extraction of mRNA from soil. Appl Environ Microbiol 76:5995–6000CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Wang Y, Hayatsu M, Fujii T (2012) Extraction of bacterial RNA from soil: challenges and solutions. Microbes Environ 27:111–121CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Kurland CG (1960) Molecular characterization of ribonucleic acid from Escherichia coli ribosomes: I. Isolation and molecular weights. J Mol Biol 2:83–91CrossRefGoogle Scholar
  9. 9.
    Chirgwin JM, Przybyla AE, MacDonald RJ et al (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294–5299CrossRefPubMedGoogle Scholar
  10. 10.
    Glišin V, Crkvenjakov R, Byus C (1974) Ribonucleic acid isolated by cesium chloride centrifugation. Biochemistry 13:2633–2637CrossRefPubMedGoogle Scholar
  11. 11.
    Patyshakuliyeva A, Mäkelä MR, Sietiö O-M et al (2014) An improved and reproducible protocol for the extraction of high quality fungal RNA from plant biomass substrates. Fungal Genet Biol 72:201–206CrossRefPubMedGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Miia R. Mäkelä
    • 1
    • 2
  • Kristiina Hildén
    • 1
  1. 1.Department of MicrobiologyUniversity of HelsinkiHelsinkiFinland
  2. 2.Fungal PhysiologyWesterdijk Fungal Biodiversity InstituteUtrechtThe Netherlands

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