Validation of Reference Genes for Transcriptional Analyses in Pleurotus ostreatus by Using Reverse Transcription-Quantitative PCR

Recently, the lignin-degrading basidiomycete Pleurotus ostreatus has become a widely used model organism for fungal genomic and transcriptomic analyses. The increasing interest in this species has led to an increasing number of studies analyzing the transcriptional regulation of multigene families t...

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Bibliographic Details
Published in:Applied and Environmental Microbiology 2015-06, Vol.81 (12), p.4120-4129
Main Authors: Castanera, Raúl, López-Varas, Leticia, Pisabarro, Antonio G, Ramírez, Lucía
Format: Article
Language:English
Subjects:
Algorithms
Basidiomycetes
Bioassays
Culture Media
Enzymes
Fermentation
Gene expression
Gene Expression Profiling
Gene Expression Regulation, Enzymologic
Gene Expression Regulation, Fungal
Genes, Essential
Genes, Fungal
Genomics
Glucose - metabolism
Laccase - genetics
Lignin
Lignin - metabolism
Methods
Mushrooms
Peroxidases - genetics
Pleurotus - enzymology
Pleurotus - genetics
Pleurotus ostreatus
Polymerase chain reaction
Real-Time Polymerase Chain Reaction - methods
Reverse Transcription
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Summary:Recently, the lignin-degrading basidiomycete Pleurotus ostreatus has become a widely used model organism for fungal genomic and transcriptomic analyses. The increasing interest in this species has led to an increasing number of studies analyzing the transcriptional regulation of multigene families that encode extracellular enzymes. Reverse transcription (RT) followed by real-time PCR is the most suitable technique for analyzing the expression of gene sets under multiple culture conditions. In this work, we tested the suitability of 13 candidate genes for their use as reference genes in P. ostreatus time course cultures for enzyme production. We applied three different statistical algorithms and obtained a combination of stable reference genes for optimal normalization of RT-quantitative PCR assays. This reference index can be used for future transcriptomic analyses and validation of transcriptome sequencing or microarray data. Moreover, we analyzed the expression patterns of a laccase and a manganese peroxidase (lacc10 and mnp3, respectively) in lignocellulose and glucose-based media using submerged, semisolid, and solid-state fermentation. By testing different normalization strategies, we demonstrate that the use of nonvalidated reference genes as internal controls leads to biased results and misinterpretations of the biological responses underlying expression changes.
ISSN:0099-2240
1098-5336
1098-6596
DOI:10.1128/AEM.00402-15