Quantitative single molecule RNA-FISH and RNase-free cell wall digestion in Neurospora crassa.
Chitinase
Fixed-cell imaging
Fungal microscopy
Quantitative cell biology
Journal
Fungal genetics and biology : FG & B
ISSN: 1096-0937
Titre abrégé: Fungal Genet Biol
Pays: United States
ID NLM: 9607601
Informations de publication
Date de publication:
11 2021
11 2021
Historique:
received:
19
07
2021
revised:
13
08
2021
accepted:
15
08
2021
pubmed:
24
8
2021
medline:
27
1
2022
entrez:
23
8
2021
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 34425213
pii: S1087-1845(21)00099-2
doi: 10.1016/j.fgb.2021.103615
pmc: PMC8463489
mid: NIHMS1738196
pii:
doi:
Substances chimiques
RNA
63231-63-0
Ribonucleases
EC 3.1.-
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Langues
eng
Sous-ensembles de citation
IM
Pagination
103615Subventions
Organisme : NIGMS NIH HHS
ID : P20 GM113132
Pays : United States
Organisme : NIGMS NIH HHS
ID : R35 GM118021
Pays : United States
Organisme : NIGMS NIH HHS
ID : R35 GM118022
Pays : United States
Organisme : NIGMS NIH HHS
ID : T32 GM008704
Pays : United States
Informations de copyright
Copyright © 2021 Elsevier Inc. All rights reserved.
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