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Selection and validation of reference genes for RT-qPCR in ophiocordyceps sinensis under different experimental conditions

Summary

Scientists studied the Chinese caterpillar mushroom (Ophiocordyceps sinensis) to find the best genes to use as reference points when measuring other gene activity. They tested sixteen potential reference genes under various stress conditions and growth stages using a technique called RT-qPCR. They found that two specific genes (18S rRNA and beta-Tubulin) were the most reliable to use as standards, which will help researchers accurately study how this medicinal mushroom responds to different conditions.

Background

Ophiocordyceps sinensis is a rare medicinal fungus in traditional Chinese herbal medicine with unique medicinal values. Accurate gene expression analysis requires stable reference genes for normalization in RT-qPCR studies. Previous studies on reference genes in macro-fungi and Cordyceps species have identified suitable internal control genes, but no such investigations are available for O. sinensis.

Objective

To identify and validate the most stable reference genes for RT-qPCR normalization in O. sinensis under different experimental conditions including abiotic stresses and developmental stages. This study evaluates sixteen candidate reference genes across different conditions to enable accurate gene expression analysis in this medicinal fungus.

Results

18S ribosomal RNA (18S rRNA) and beta-Tubulin (β-TUB) showed the highest expression stability across all conditions, while glutathione hydrolase proenzyme (GGT) and Phosphoglucose isomerase (PGI) showed the least stability. Paired variation values indicated that two genes were sufficient for accurate normalization. Optimal reference gene combinations varied by condition: β-TUB and UBQ for primordia development, PGM with EF1-α for morphologic mycelium stages, and 18S rRNA with UBQ for different stresses.

Conclusion

18S rRNA and β-TUB are recommended as the most reliable reference genes for RT-qPCR normalization in O. sinensis across various experimental conditions. Condition-specific optimal reference gene combinations have been identified to ensure accurate gene expression analysis. These results provide essential standardization for future molecular studies of this valuable medicinal fungus.
  • Published in:PLoS One,
  • Study Type:Experimental validation study,
  • Source: 10.1371/journal.pone.0287882, PMID: 38319940
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