title: Validation of Reference Genes for Transcriptional Analyses in Pleurotus ostreatus by Using Reverse Transcription-Quantitative PCR.
abstract: 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.
authors: Genetics and Microbiology Research Group, Department of Agrarian Production, Public University of Navarre, Pamplona, Navarre, Spain.
authors: Raúl
authors: R
authors: Castanera
authors: Genetics and Microbiology Research Group, Department of Agrarian Production, Public University of Navarre, Pamplona, Navarre, Spain.
authors: Leticia
authors: L
authors: López-Varas
authors: Genetics and Microbiology Research Group, Department of Agrarian Production, Public University of Navarre, Pamplona, Navarre, Spain.
authors: Antonio G
authors: AG
authors: Pisabarro
authors: Genetics and Microbiology Research Group, Department of Agrarian Production, Public University of Navarre, Pamplona, Navarre, Spain lramirez@unavarra.es.
authors: Lucía
authors: L
authors: Ramírez
mesh_terms: D000465:Algorithms; D003470:Culture Media; D005285:Fermentation; D020869:Gene Expression Profiling; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D020043:Genes, Essential; D005800:Genes, Fungal; D005947:Glucose / Q000378:metabolism; D042845:Laccase / Q000235:genetics; D008031:Lignin / Q000378:metabolism; D010544:Peroxidases / Q000235:genetics; D020076:Pleurotus / Q000201:enzymology* / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D048348:Reverse Transcription
chemical_list: D003470:Culture Media; C036909:lignocellulose; D008031:Lignin; D042845:Laccase; D010544:Peroxidases; C051129:manganese peroxidase; D005947:Glucose