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Isolation of total RNA from dermatophytes
S Rezaie1, M Pourmojib, E Tschachler
1Division of Immunology, Allergy and Infectious Diseases, University of Vienna Medical School, Austria.
Mycoses
|February 19, 2000
Summary
This study presents a reliable method for isolating high-quality total RNA from Trichophyton rubrum, a common fungal pathogen. The procedure ensures excellent messenger RNA (mRNA) integrity, applicable to other dermatophytes.
Area of Science:
- Mycology
- Molecular Biology
- Biochemistry
Background:
- Dermatophytes are pathogenic fungi causing superficial infections.
- Efficient RNA extraction is crucial for studying gene expression in fungi.
- Previous methods for RNA isolation from Trichophyton species were limited.
Purpose of the Study:
- To develop and validate a robust method for total RNA preparation from Trichophyton rubrum.
- To ensure the quality and integrity of the extracted RNA for downstream applications.
- To assess the general applicability of the method across different dermatophyte species.
Main Methods:
- Cultivation of Trichophyton rubrum in liquid medium to obtain sufficient mycelial biomass.
- Homogenization of mycelia in guanidine isothiocyanate buffer after grinding in liquid nitrogen.
- Purification of total RNA using ultracentrifugation through a cesium chloride gradient.
Main Results:
- Isolation of high-quality total RNA with distinct ribosomal RNA (rRNA) bands (approx. 3.36 and 1.82 kb).
- Confirmation of intact messenger RNA (mRNA) integrity via Northern blot hybridization using beta-actin cDNA.
- Successful RNA extraction from Trichophyton mentagrophytes and Microsporum canis, indicating method versatility.
Conclusions:
- The described method provides a reliable means for preparing high-quality total RNA from Trichophyton rubrum.
- The procedure yields RNA suitable for molecular analyses, including Northern blotting.
- This technique is broadly applicable to other medically important dermatophyte species.