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Quantitative single molecule RNA-FISH and RNase-free cell wall digestion in Neurospora crassa.

Publication ,  Journal Article
Bartholomai, BM; Gladfelter, AS; Loros, JJ; Dunlap, JC
Published in: Fungal Genet Biol
November 2021

Single molecule RNA-FISH (smFISH) is a valuable tool for analysis of mRNA spatial patterning in fixed cells that is underutilized in filamentous fungi. A primary complication for fixed-cell imaging in filamentous fungi is the need for enzymatic cell wall permeabilization, which is compounded by considerable variability in cell wall composition between species. smFISH adds another layer of complexity due to a requirement for RNase free conditions. Here, we describe the cloning, expression, and purification of a chitinase suitable for supplementation of a commercially available RNase-free enzyme preparation for efficient permeabilization of the Neurospora cell wall. We further provide a method for smFISH in Neurospora which includes a tool for generating numerical data from images that can be used in downstream customized analysis protocols.

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Published In

Fungal Genet Biol

DOI

EISSN

1096-0937

Publication Date

November 2021

Volume

156

Start / End Page

103615

Location

United States

Related Subject Headings

  • Ribonucleases
  • RNA
  • Neurospora crassa
  • Microbiology
  • Digestion
  • Cell Wall
  • 3108 Plant biology
  • 3107 Microbiology
  • 3105 Genetics
  • 0607 Plant Biology
 

Citation

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Bartholomai, B. M., Gladfelter, A. S., Loros, J. J., & Dunlap, J. C. (2021). Quantitative single molecule RNA-FISH and RNase-free cell wall digestion in Neurospora crassa. Fungal Genet Biol, 156, 103615. https://doi.org/10.1016/j.fgb.2021.103615
Bartholomai, Bradley M., Amy S. Gladfelter, Jennifer J. Loros, and Jay C. Dunlap. “Quantitative single molecule RNA-FISH and RNase-free cell wall digestion in Neurospora crassa.Fungal Genet Biol 156 (November 2021): 103615. https://doi.org/10.1016/j.fgb.2021.103615.
Bartholomai BM, Gladfelter AS, Loros JJ, Dunlap JC. Quantitative single molecule RNA-FISH and RNase-free cell wall digestion in Neurospora crassa. Fungal Genet Biol. 2021 Nov;156:103615.
Bartholomai, Bradley M., et al. “Quantitative single molecule RNA-FISH and RNase-free cell wall digestion in Neurospora crassa.Fungal Genet Biol, vol. 156, Nov. 2021, p. 103615. Pubmed, doi:10.1016/j.fgb.2021.103615.
Bartholomai BM, Gladfelter AS, Loros JJ, Dunlap JC. Quantitative single molecule RNA-FISH and RNase-free cell wall digestion in Neurospora crassa. Fungal Genet Biol. 2021 Nov;156:103615.
Journal cover image

Published In

Fungal Genet Biol

DOI

EISSN

1096-0937

Publication Date

November 2021

Volume

156

Start / End Page

103615

Location

United States

Related Subject Headings

  • Ribonucleases
  • RNA
  • Neurospora crassa
  • Microbiology
  • Digestion
  • Cell Wall
  • 3108 Plant biology
  • 3107 Microbiology
  • 3105 Genetics
  • 0607 Plant Biology