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

IF: 3.883

Cited by: 2

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.

Keywords

smFISH

Gene Expression

Chitinase

Fixed-cell imaging

Fungal microscopy

Quantitative cell biology

MeSH terms

Cell Wall

Digestion

Neurospora crassa

RNA

Ribonucleases

Authors

Bartholomai, Bradley M

Gladfelter, Amy S

Loros, Jennifer J

Dunlap, Jay C

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