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Optimized Protocol for RNA Isolation from Penicillium spp. and Aspergillus fumigatus Strains

Summary

Scientists developed an improved method for extracting RNA from common mold fungi like Penicillium and Aspergillus. The study compared two extraction techniques and found that using a mechanical bead-beater device combined with a chemical solvent called chloroform produced the best results. This optimized method yields high-quality RNA suitable for studying gene expression in these fungi and can be easily used in regular laboratory settings.

Background

Efficient RNA isolation from filamentous fungi is crucial for gene expression studies but poses significant technical challenges due to robust cell walls and RNA susceptibility to ribonuclease degradation. The complex structure of fungal cells, which includes cell walls composed of chitin and glucans, makes RNA isolation particularly difficult compared to other organisms.

Objective

To compare the effectiveness of two RNA isolation protocols for four species of filamentous fungi: Penicillium crustosum, Penicillium rubens, Penicillium griseofulvum, and Aspergillus fumigatus, varying in mechanical disruption methods (bead-beating versus manual vortexing).

Results

Protocol 1 (bead-beater method) yielded significantly higher RNA quantities (30-96 µg/g dry biomass in Penicillium species, up to 52 µg/g in A. fumigatus) with better purity and integrity (A260/A280 and A260/A230 ratios exceeding 2 and 1.8 respectively). Protocol 2 yielded approximately 2.2 times lower RNA concentrations with insufficient purity for cDNA synthesis. Chloroform extraction in Protocol 1 effectively separated contaminants including DNA, proteins, and polysaccharides.

Conclusion

The optimized protocol combining mechanical lysis with a bead-beater and RNA extraction with chloroform is highly efficient for filamentous fungi, producing high-quality RNA suitable for downstream applications such as cDNA synthesis and transcriptome analysis. This method is practical for routine laboratories handling large numbers of fungal samples.
  • Published in:Current Issues in Molecular Biology,
  • Study Type:Comparative Study,
  • Source: PMID: 39590372, DOI: 10.3390/cimb46110778
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