Quantitative single molecule RNA-FISH and RNase-free cell wall digestion in Neurospora crassa.
Fungal Genet Biol
; 156: 103615, 2021 11.
Article
en En
| MEDLINE
| ID: mdl-34425213
ABSTRACT
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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Neurospora crassa
Idioma:
En
Revista:
Fungal Genet Biol
Asunto de la revista:
GENETICA
/
MICROBIOLOGIA
Año:
2021
Tipo del documento:
Article
País de afiliación:
Estados Unidos