Finding Stable Genes for Genetic Testing Using RNA Data

Greg Howard
10th May, 2025

Finding Stable Genes for Genetic Testing Using RNA Data

Wine-cap Stropharia (Stropharia rugosoannulata)

Photo adapted from: Annie Weissman / CC BY (Source)

Key Findings

  • In China, scientists identified UBP as the most reliable reference gene for studying the wine-cap mushroom’s gene activity
  • Traditional benchmark genes like GAPDH were found unstable, making them unsuitable for accurate gene measurements
  • The new reference genes enhance future research on the mushroom’s growth and environmental benefits
Quantitative real-time PCR (qRT-PCR) is a widely used technique for measuring gene expression, allowing scientists to understand how genes behave under different conditions. However, to obtain accurate and reliable results, it's essential to use stable reference genes. These genes act as benchmarks, ensuring that the measurements of target genes are consistent and meaningful. Selecting the right reference genes is crucial because unstable ones can lead to incorrect conclusions. Stropharia rugosoannulata, commonly known as the wine-cap Stropharia mushroom, is not only a popular edible mushroom but also one of the top ten internationally traded mushrooms. Its significance in both culinary and ecological fields has spurred extensive research. Previous studies have explored its genome, revealing important insights into its capabilities for lignin degradation and bioremediation[2][3]. Despite these advancements, molecular studies, including gene expression analyses, have been limited due to the scarcity of stable reference genes specific to this species. A recent study conducted by researchers at the All China Federation of Supply & Marketing Cooperatives, China, addresses this gap[1]. The researchers aimed to identify and validate stable reference genes for qRT-PCR in S. rugosoannulata, enhancing the reliability of gene expression studies in this mushroom. To achieve this, they selected six novel candidate reference genes from the mushroom's transcriptome and included four traditional reference genes that were previously considered stable. The study employed three widely recognized software tools—geNorm, NormFinder, and BestKeeper—to evaluate the stability of the ten candidate reference genes across various developmental stages of both red and yellow varieties of S. rugosoannulata. These tools analyze gene expression data to determine which genes maintain consistent expression levels under different experimental conditions. The final ranking of the reference genes was determined using RefFinder, a comprehensive tool that integrates the results from the three software packages to provide a more accurate assessment. The findings revealed that the gene UBP exhibited the highest stability across all tested developmental stages, making it the most reliable reference gene for future qRT-PCR studies in S. rugosoannulata. In contrast, RPB2 and GAPDH, two of the traditional reference genes, showed the least stability, indicating that they may not be suitable for normalization in this context. Notably, the six novel reference genes outperformed the traditional ones in terms of stability, highlighting the importance of species-specific validation. This advancement is particularly significant given the previous genomic studies on S. rugosoannulata. For instance, researchers have sequenced its genome, uncovering genes involved in lignin degradation and bioremediation[2][3]. These findings have laid the groundwork for understanding the mushroom's biological functions and its potential applications in environmental sustainability. However, without reliable reference genes, accurately measuring gene expression related to these functions has been challenging. The current study bridges this gap by providing validated reference genes, thereby facilitating more precise and reliable molecular research. Moreover, this research builds on methodologies used in other species, such as radish, where reference gene stability has also been thoroughly investigated[4]. By adapting and refining these approaches for S. rugosoannulata, the study ensures that the reference genes selected are tailored to the specific genetic and developmental contexts of the mushroom. The implications of this study extend beyond basic research. With stable reference genes in place, scientists can more effectively explore gene expression patterns related to S. rugosoannulata's growth, development, and environmental interactions. This can lead to improved cultivation techniques, enhanced nutritional profiles, and better utilization of the mushroom in bioremediation projects. Additionally, understanding gene expression with greater accuracy can support breeding programs aimed at developing strains with desirable traits, such as increased yield or enhanced degradation capabilities. In conclusion, the identification of stable reference genes for qRT-PCR in S. rugosoannulata represents a significant step forward in mushroom research. By ensuring reliable normalization of gene expression data, this study empowers scientists to delve deeper into the molecular mechanisms that drive the mushroom's valuable properties. As a result, the wine-cap Stropharia mushroom can continue to be a focus of both culinary and environmental research, supported by robust and accurate genetic analysis.

GeneticsBiochemMycology

References

Main Study

1) Identification of stable reference genes for qRT-PCR in Stropharia rugosoannulata using mRNA-sequencing data

Published 7th May, 2025

https://doi.org/10.1371/journal.pone.0323272

Related Studies

2) Whole Genome Sequence of an Edible Mushroom Stropharia rugosoannulata (Daqiugaigu).

https://doi.org/10.3390/jof8020099

3) Genomic Analysis of Stropharia rugosoannulata Reveals Its Nutritional Strategy and Application Potential in Bioremediation.

https://doi.org/10.3390/jof8020162

4) Evaluation of reference genes for gene expression studies in radish (Raphanus sativus L.) using quantitative real-time PCR.

https://doi.org/10.1016/j.bbrc.2012.06.119


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