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

Summary

Researchers developed an improved method for extracting RNA from common mold species like Penicillium and Aspergillus fumigatus. The new protocol uses physical shaking with beads and chemical extraction to break open fungal cells and isolate high-quality RNA. This method produces significantly more usable RNA than previous approaches and can be easily applied in laboratories working with many fungal samples.

Background

RNA isolation from filamentous fungi is essential for gene expression studies but presents significant technical challenges due to robust cell walls and RNA susceptibility to degradation by ribonucleases. Various RNA isolation techniques have been developed including mechanical, chemical, and enzymatic methods, often used in combination to optimize efficiency and quality.

Objective

This study compared the effectiveness of two RNA isolation protocols for four filamentous fungal species: Penicillium crustosum, P. rubens, P. griseofulvum, and Aspergillus fumigatus. Both protocols utilized Fenzol Plus but differed in mechanical disruption methods: bead-beating versus manual vortexing.

Results

Protocol 1 yielded significantly higher RNA quantities (30-96 µg/g in Penicillium species, up to 52 µg/g in A. fumigatus) with better purity and integrity, demonstrated by higher A260/A280 and A260/A230 ratios. Protocol 2 yielded approximately 2.2 times lower RNA amounts with insufficient purity for further analysis. The use of chloroform in Protocol 1 effectively removed DNA, proteins, and polysaccharides.

Conclusion

The bead-beater method combined with chloroform extraction proved optimal for RNA isolation from Penicillium and Aspergillus species, yielding high-quality RNA suitable for cDNA synthesis and transcriptome analysis. This protocol is efficient, practical for routine laboratory use with large sample loads, and represents a robust method for downstream molecular applications.
  • Published in:Current Issues in Molecular Biology,
  • Study Type:Experimental Comparison Study,
  • Source: PMID: 39590372, DOI: 10.3390/cimb46110778
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