Skip to main content

Isolation of Total RNA From Neurospora Mycelium

  • Protocol
Circadian Rhythms

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 362))

Abstract

In filamentous fungi, including the model organism Neurospora crassa, plentiful biological tissue from which RNA can be extracted may be obtained by allowing fungal spores to germinate and form a mycelium in liquid culture. The mycelium constitutes a mosaic of multinuclear, tubular filaments known as hyphae or mycelia. In general, when exposed to air, fungal hyphae quickly start to develop spores, which are often colorful. However, when submerged in liquid under rapid agitation large amounts of vegetatively growing mycelium can be obtained, which can be easily harvested by means of filtration. To preserve the physiological state of the culture, the mycelium is snap-frozen, and then to free its contents, the mycelium is ground under liquid nitrogen to break all hyphal structures. Here a method to extract high-quality total RNA from Neurospora mycelium using TRIzol® reagent is described.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 219.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Sela, M., Anfinsen, C. B.. and Harrington, W. F. (1957) The correlation of ribonuclease activity with specific aspects of tertiary structure. Biochim. Biophys. Acta 26, 502–512.

    Article  CAS  PubMed  Google Scholar 

  2. Cox, R. A. (1968) The use of guanidinium chloride in the isolation of nucleic acids. Methods Enzymol. 12B, 120–129.

    Article  Google Scholar 

  3. Nozaki, Y., and Tanford, C. (1970) The solubility of amino acids, diglycine, and triglycine in aqueous guanidine hydrochloride solutions. J. Biol. Chem. 245, 1648–1652.

    CAS  PubMed  Google Scholar 

  4. Gordon, J. A. (1972) Denaturation of globular proteins. Interaction of guanidinium salts with three proteins. Biochemistry 11, 1862–1870.

    Article  CAS  PubMed  Google Scholar 

  5. Chomczynski, P., and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159.

    Article  CAS  PubMed  Google Scholar 

  6. Chomczynski, P. (1993) A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. Biotechniques 15, 532–537.

    CAS  PubMed  Google Scholar 

  7. TRIzol® Reagent. Invitrogen. http://www.invitrogen.com/content.cfm?pageid=469.

  8. Springer, M. L. (1993) Genetic control of fungal differentiation: the three sporulation pathways of Neurospora crassa. BioEssays 15, 365–374.

    Article  CAS  PubMed  Google Scholar 

  9. Davis, R. H. (2000) Neurospora: contributions of a model organism. Oxford University Press, New York.

    Google Scholar 

  10. Nakashima, H. (1981) A liquid culture system for the biochemical analysis of the circadian clock of Neurospora. Plant Cell Physiol. 22, 231–238.

    CAS  Google Scholar 

  11. Perlman, J., Nakashima, H., and Feldman, J. (1981) Assay and characteristics of circadian rhythmicity in liquid cultures of Neurospora crassa. Plant Physiol. 67, 404–407.

    Article  CAS  PubMed  Google Scholar 

  12. Loros, J., and Dunlap, J. C. (1991) Neurospora crassa clock-controlled genes are regulated at the level of transcription. Mol. Cell. Biol. 11, 558–563.

    CAS  PubMed  Google Scholar 

  13. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

    Google Scholar 

  14. Vogel, H. J. (1956) A convenient growth medium for Neurospora (Medium N). Microbiol. Genet. Bull. 13, 42–43.

    Google Scholar 

  15. Davis, R. H., and de Serres, F. J. (1970) Genetic and microbial research techniques for Neurospora crassa. Methods Enzymol. 17A, 79–143.

    Article  Google Scholar 

  16. Pittendrigh, C. S., Bruce, V. G., Rosenzweig, N. S., and Rubin, M. L. (1959) A biological clock in Neurospora. Nature 184, 169–170.

    Article  Google Scholar 

  17. Francis, C. D., and Sargent, M. L. (1979) Effects of temperature perturbations on circadian conidiation in Neurospora. Plant Physiol. 64, 1000–1004.

    Article  CAS  PubMed  Google Scholar 

  18. Crosthwaite, S. K., Loros, J. J., and Dunlap, J. C. (1995) Light-induced resetting of a circadian clock mediated by a rapid increase in frequency transcript. Cell 81, 1003–1012.

    Article  CAS  PubMed  Google Scholar 

  19. Kramer, C., Loros, J. J., Dunlap, J. C., and Crosthwaite, S. K. (2003) Role for antisense RNA in regulating circadian clock function in Neurospora crassa. Nature 421, 948–952.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2007 Humana Press Inc.

About this protocol

Cite this protocol

Kramer, C. (2007). Isolation of Total RNA From Neurospora Mycelium. In: Rosato, E. (eds) Circadian Rhythms. Methods in Molecular Biology™, vol 362. Humana Press. https://doi.org/10.1007/978-1-59745-257-1_19

Download citation

  • DOI: https://doi.org/10.1007/978-1-59745-257-1_19

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-417-3

  • Online ISBN: 978-1-59745-257-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics