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.
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References
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.
Cox, R. A. (1968) The use of guanidinium chloride in the isolation of nucleic acids. Methods Enzymol. 12B, 120–129.
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.
Gordon, J. A. (1972) Denaturation of globular proteins. Interaction of guanidinium salts with three proteins. Biochemistry 11, 1862–1870.
Chomczynski, P., and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159.
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.
TRIzol® Reagent. Invitrogen. http://www.invitrogen.com/content.cfm?pageid=469.
Springer, M. L. (1993) Genetic control of fungal differentiation: the three sporulation pathways of Neurospora crassa. BioEssays 15, 365–374.
Davis, R. H. (2000) Neurospora: contributions of a model organism. Oxford University Press, New York.
Nakashima, H. (1981) A liquid culture system for the biochemical analysis of the circadian clock of Neurospora. Plant Cell Physiol. 22, 231–238.
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.
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.
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.
Vogel, H. J. (1956) A convenient growth medium for Neurospora (Medium N). Microbiol. Genet. Bull. 13, 42–43.
Davis, R. H., and de Serres, F. J. (1970) Genetic and microbial research techniques for Neurospora crassa. Methods Enzymol. 17A, 79–143.
Pittendrigh, C. S., Bruce, V. G., Rosenzweig, N. S., and Rubin, M. L. (1959) A biological clock in Neurospora. Nature 184, 169–170.
Francis, C. D., and Sargent, M. L. (1979) Effects of temperature perturbations on circadian conidiation in Neurospora. Plant Physiol. 64, 1000–1004.
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.
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.
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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
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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
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