<i>Act1</i> out of Action: Identifying Reliable Reference Genes in <i>Trichoderma reesei</i> for Gene Expression Analysis
<i>Trichoderma reesei</i> is a well-established industrial enzyme producer and has been the subject of extensive research for various applications. The basis of many research studies is the analysis of gene expression, specifically with RT-qPCR, which requires stable reference genes for...
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| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2025-05-01
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| Series: | Journal of Fungi |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2309-608X/11/5/396 |
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| Summary: | <i>Trichoderma reesei</i> is a well-established industrial enzyme producer and has been the subject of extensive research for various applications. The basis of many research studies is the analysis of gene expression, specifically with RT-qPCR, which requires stable reference genes for normalization to yield reliable results. Yet the commonly used reference genes, <i>act1</i> and <i>sar1</i>, were initially chosen based on reports from the literature rather than systematic validation, raising concerns about their stability. Thus, properly evaluated reference genes for <i>T. reesei</i> are lacking. In this study, five potentially new reference genes were identified by analyzing publicly available transcriptome datasets of the <i>T. reesei</i> strains QM6a and Rut-C30. Their expression stability was then evaluated under relevant cultivation conditions using RT-qPCR and analyzed with RefFinder. The two most stable candidate reference genes were further validated by normalizing the expression of the well-characterized gene <i>cbh1</i> and comparing the results to those obtained using <i>act1</i> and <i>sar1</i>. Additionally, <i>act1</i> and <i>sar1</i> were normalized against the new reference genes to assess the variability in their expression. All five new reference genes exhibited a more stable expression than <i>act1</i> and <i>sar1</i>. Both in silico and RT-qPCR analysis ranked the so far uncharacterized gene, <i>bzp1</i>, as the most stable. Further, we found that <i>act1</i> and <i>sar1</i> have strain- and condition-dependent expression variability, suggesting that they are unsuitable as universal reference genes in <i>T. reesei</i>. Based on these results, we propose to use the combination of <i>bzp1</i> and <i>tpc1</i> for the normalization in RT-qPCR analysis instead of <i>act1</i> and <i>sar1</i>. |
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| ISSN: | 2309-608X |