Identification and evaluation of reference genes for qRT-PCR normalization in Ganoderma lucidum

Area of Science:

• Mycology
• Molecular Biology
• Biotechnology

Background:

• Quantitative real-time reverse transcription PCR (qRT-PCR) is crucial for gene expression analysis.
• Accurate qRT-PCR relies on stable reference genes for normalization.
• Ganoderma lucidum, a medicinal mushroom, requires validated reference genes for reliable research.

Purpose of the Study:

• To systematically evaluate the expression stability of candidate reference genes in Ganoderma lucidum.
• To identify the most suitable reference genes for normalizing qRT-PCR data across different experimental conditions.
• To establish a foundation for accurate gene expression studies in Ganoderma lucidum.

Main Methods:

• Selection and manual curation of 10 potential reference genes from Ganoderma lucidum genomic data.
• Primer design adhering to strict criteria for qRT-PCR.
• Assessment of gene expression stability using qRT-PCR and statistical algorithms (geNorm, NormFinder, BestKeeper, RefFinder).

Main Results:

• PP2A exhibited the most stable expression under varying fermentation conditions.
• RPL4 demonstrated the highest stability across different tissue types.
• RPL4, PP2A, and β-tubulin were identified as the most reliable reference genes for the entire dataset.

Conclusions:

• The study provides validated reference genes for accurate qRT-PCR in Ganoderma lucidum.
• PP2A and RPL4 are recommended for specific experimental contexts (fermentation and tissues, respectively).
• The findings support robust gene expression analysis in Ganoderma lucidum research.