Abstract
Aspergillus is a genus of filamentous fungi with vast geographic and ecological distributions. Species within this genus are clinically, agriculturally and biotechnologically relevant, leading to increasing interest in elucidating gene expression dynamics of key metabolic and physiological processes. Reverse-transcription quantitative Polymerase Chain Reaction (RT-qPCR) is a sensitive and specific method of quantifying gene expression. A crucial step for comparing RT-qPCR results between strains and experimental conditions is normalisation to experimentally validated reference gene(s). In this review, we provide a critical analysis of current reference gene selection and validation practices for RT-qPCR gene expression analyses of Aspergillus. Of 90 primary research articles obtained through our PubMed query, 17 experimentally validated the reference gene(s) used. Twenty reference genes were used across the 90 studies, with beta-tubulin being the most used reference gene, followed by actin, 18S rRNA and glyceraldehyde 3-phosphate dehydrogenase. Sixteen of the 90 studies used multiple reference genes for normalisation. Failing to experimentally validate the stability of reference genes can lead to conflicting results, as was the case for four studies. Overall, our review highlights the need to experimentally validate reference genes in RT-qPCR studies of Aspergillus.
Highlights
The ascomycete genus Aspergillus is among the first described filamentous fungi, dating back to 1729, as recorded by Pier Antonio Micheli, an Italian biologist and priest
We describe the reference genes that have been used for normalising gene expressions in Reverse-transcription quantitative Polymerase Chain Reaction (RT-qPCR) analyses for species in the genus Aspergillus
antimicrobial peptides (AMPs) tested, two (77-3 and D4E1) were the most effective inhibitors of expression [55]. These RT-qPCR studies using beta-tubulin as a reference gene helped reveal the general conditions associated with increases, and decreases in aflatoxin production by A. flavus and A. parasiticus
Summary
The ascomycete genus Aspergillus is among the first described filamentous fungi, dating back to 1729, as recorded by Pier Antonio Micheli, an Italian biologist and priest. Aspergillus fungi are broadly distributed across the globe, and are found in diverse ecological niches such as soil, composts, water, buildings, air, and in or on plants [4] Species in this genus have significant impacts on many fields, including biotechnology (e.g., antibiotics production) [5], fermented food production [6], food safety (e.g., mycotoxin production and food contamination) [7] and human health [2]. Several approaches and techniques have been developed to monitor and quantify gene expression These techniques include Northern blotting, microarray hybridisation, high throughput transcriptome sequencing and reverse-transcription quantitative polymerase chain reaction (RT-qPCR). We describe the reference genes that have been used for normalising gene expressions in RT-qPCR analyses for species in the genus Aspergillus. We discuss the potential areas of improvements for selecting robust reference genes
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