Tracking the best reference genes for RT-qPCR data normalization in filamentous fungi

BMC Genomics
Agustina LlanosJean-Luc Parrou

Abstract

A critical step in the RT-qPCR workflow for studying gene expression is data normalization, one of the strategies being the use of reference genes. This study aimed to identify and validate a selection of reference genes for relative quantification in Talaromyces versatilis, a relevant industrial filamentous fungus. Beyond T. versatilis, this study also aimed to propose reference genes that are applicable more widely for RT-qPCR data normalization in filamentous fungi. A selection of stable, potential reference genes was carried out in silico from RNA-seq based transcriptomic data obtained from T. versatilis. A dozen functionally unrelated candidate genes were analysed by RT-qPCR assays over more than 30 relevant culture conditions. By using geNorm, we showed that most of these candidate genes had stable transcript levels in most of the conditions, from growth environments to conidial germination. The overall robustness of these genes was explored further by showing that any combination of 3 of them led to minimal normalization bias. To extend the relevance of the study beyond T. versatilis, we challenged their stability together with sixteen other classically used genes such as β-tubulin or actin, in a representative sample of...Continue Reading

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Datasets Mentioned

BETA
GSE44648
GSE30031
GSE30579
GSE18851
GSE44100
GSE43163
GSE33683

Methods Mentioned

BETA
PCR
RNA-seq
Assay
RNA seq
GTPase

Software Mentioned

BLAST
CFX Manager
geNorm VBA
Normfinder
Bestkeeper
EQUAL
STATGRAPHICS Centurion
geNorm
GEO
RefGenes

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