Abstract
The selection of appropriate internal control genes (ICGs) is a crucial step in the normalization of real-time quantitative PCR (RT-qPCR) data. Housekeeping genes are habitually selected for this purpose, despite accumulating evidence on their instability. We screened for novel, robust ICGs in the mushroom forming fungus Volvariella volvacea. Nine commonly used and five newly selected ICGs were evaluated for expression stability using RT-qPCR data in eight different stages of the life cycle of V. volvacea. Three different algorithms consistently determined that three novel ICGs (SPRYp, Ras and Vps26) exhibited the highest expression stability in V. volvacea. Subsequent analysis of ICGs in twenty-four expression profiles from nine filamentous fungi revealed that Ras was the most stable ICG amongst the Basidiomycetous samples, followed by SPRYp, Vps26 and ACTB. Vps26 was expressed most stably within the analyzed data of Ascomycetes, followed by HH3 and β-TUB. No ICG was universally stable for all fungal species, or for all experimental conditions within a species. Ultimately, the choice of an ICG will depend on a specific set of experiments. This study provides novel, robust ICGs for Basidiomycetes and Ascomycetes. Together with the presented guiding principles, this enables the efficient selection of suitable ICGs for RT-qPCR.
Highlights
Real-time quantitative PCR (RT-qPCR) has emerged as a powerful and popular tool for rapid and accurate assessment of changes in gene expression[1,2]
In this study we identified known and novel internal control genes (ICGs) in the mushroom forming fungus Volvariella volvacea, and assessed their suitability in various filamentous fungi
Nine housekeeping genes that are traditionally used as ICGs were identified in the genome of V. volvacea and confirmed by BLAST in NCBI: 18S rRNA (18S), β-actin (ACTB), cyclophilin (CYP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone H3 (HH3), tubulin (α-TUB, β-TUB1, β-TUB2) and ubiquitin (UBQ)
Summary
Real-time quantitative PCR (RT-qPCR) has emerged as a powerful and popular tool for rapid and accurate assessment of changes in gene expression[1,2]. Existing reports on fungal ICGs mainly focus on relative expression stability[16,17,18], and ignore absolute expression levels. These studies mostly involve traditional housekeeping genes in Ascomycetes[10,14,16,17,18,19] and except for Phakopsora pachyrhizi[20] and Pleurotus ostreatus[21]; little information is available on ICGs in Basidomycetes. In this study we identified known and novel ICGs in the mushroom forming fungus Volvariella volvacea, and assessed their suitability in various filamentous fungi. The selected V. volvacea ICGs were examined for their stability in other filamentous fungi
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