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Identification of Novel and Robust Internal Control Genes from Volvariella volvacea that are Suitable for RT-qPCR in Filamentous Fungi

Summary

This research identified better reference genes for measuring gene activity in fungi. Reference genes are essential tools that scientists use to study how other genes work in organisms. The study found three new reference genes that work better than traditionally used ones, especially in mushroom-forming fungi. This discovery helps make genetic research in fungi more accurate and reliable. Impacts on everyday life: • Enables more accurate research on mushroom production for food industry • Helps improve understanding of how fungi grow and develop • Contributes to better methods for studying genes in organisms • Could lead to improvements in mushroom farming techniques • Advances our fundamental knowledge of fungal biology

Background

Real-time quantitative PCR (RT-qPCR) requires stable internal control genes (ICGs) for accurate normalization of gene expression data. Traditionally used housekeeping genes often show irregular expression under different conditions, making them unreliable as ICGs. The selection of appropriate ICGs with consistent expression levels is therefore crucial for reliable RT-qPCR analysis.

Objective

To identify and validate novel, robust internal control genes in the mushroom forming fungus Volvariella volvacea that could serve as reliable reference genes for RT-qPCR analysis in filamentous fungi. The study aimed to evaluate both traditional housekeeping genes and newly identified candidates across different developmental stages and experimental conditions.

Results

Three novel ICGs (SPRYp, Ras and Vps26) showed consistently higher expression stability compared to traditional housekeeping genes in V. volvacea. Analysis of expression data from other fungi revealed that Ras was the most stable ICG among Basidiomycetes, followed by SPRYp and Vps26. In Ascomycetes, Vps26 showed the highest stability, followed by histone H3 and β-tubulin. No single ICG was universally stable across all fungal species or experimental conditions.

Conclusion

The study identified three novel ICGs that showed superior stability compared to traditionally used housekeeping genes in filamentous fungi. While no universal ICG was found suitable for all fungi or conditions, the newly identified genes provide robust alternatives for RT-qPCR normalization. The study emphasizes the importance of validating ICGs under specific experimental conditions and provides guidelines for selecting appropriate reference genes.
  • Published in:Scientific Reports,
  • Study Type:Laboratory Research,
  • Source: 10.1038/srep29236
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