Selection of reference genes in Thermothelomyces fergusii: insights from genomic and expression analysis in response to different carbon sources.

Background: RNA expression analysis using reverse transcription qPCR requires reference genes for accurate data normalization. However, some reference genes can show significant variability in specific biological samples, raising concerns about the reliability of this method. This study assessed the expression levels of 12 reference genes in Thermothelomyces fergusii during spore and mycelium cultivation under different carbon source media (avicel, cellobiose, and glucose) using RT-qPCR. T. fergusii is a thermophilic fungus known for its abundance of cellulase and industrial applications. Quantifying fungal gene expression using the RT-qPCR method is essential for investigating the molecular biology of T. fergusii.

Results: The stability of 12 reference genes in T. fergusii was evaluated using RefFinder, geNorm, NormFinder, BestKeeper, and delta Ct techniques. The robustness of the chosen reference gene was verified using the LPMO AA9-4 gene. Analysis of the different methods showed variations in the results of reference genes under different sugar treatments. Consistently, EF1, GAPDH, and 18 S-3 were identified as reliable reference genes by the geometric mean of ranking values from Pearson correlation coefficient (r) analysis of pooled data, ranking values of RefFinder in pooled data, and NormFinder of pooled data. However, BT-3, ACT-3, and 18 S-2 exhibited inconsistency as reference genes.

Conclusion: These results emphasize the importance of verifying reference genes in specific experimental models and provide valuable recommendations for selecting reliable reference genes in RT-qPCR. These findings have broad applicability for gene expression investigations involving T. fergusii.