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Reference gene identification for reliable normalisation of quantitative RT-PCR data in Setaria viridis2.09 MB
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Journal article
Reference gene identification for reliable normalisation of quantitative RT-PCR data in Setaria viridis
Plant Methods, Vol.14(1), pp.1-12
2018
PMCID: PMC5861610
PMID: 29581727
Abstract
Background: Quantitative real-time polymerase chain reaction (RT-qPCR) is the key platform for the quantitative analysis of gene expression in a wide range of experimental systems and conditions. However, the accuracy and reproducibility of gene expression quantification via RT-qPCR is entirely dependent on the identification of reliable reference genes for data normalisation. Green foxtail (Setaria viridis) has recently been proposed as a potential experimental model for the study of C-4 photosynthesis and is closely related to many economically important crop species of the Panicoideae subfamily of grasses, including Zea mays (maize), Sorghum bicolor (sorghum) and Sacchurum officinarum (sugarcane). Setaria viridis (Accession 10) possesses a number of key traits as an experimental model, namely; (i) a small sized, sequenced and well annotated genome; (ii) short stature and generation time; (iii) prolific seed production, and; (iv) is amendable to Agrobacterium tumefaciens-mediated transformation. There is currently however, a lack of reference gene expression information for Setaria viridis (S. viridis). We therefore aimed to identify a cohort of suitable S. viridis reference genes for accurate and reliable normalisation of S. viridis RT-qPCR expression data.
Results: Eleven putative candidate reference genes were identified and examined across thirteen different S. viridis tissues. Of these, the geNorm and NormFinder analysis software identified SERINE/THERONINE-PROTEIN PHOSPHATASE 2A (PP2A), 5'-ADENYLYLSULFATE REDUCTASE 6 (ASPR6) and DUAL SPECIFICITY PHOSPHATASE (DUSP) as the most suitable combination of reference genes for the accurate and reliable normalisation of S. viridis RT-qPCR expression data. To demonstrate the suitability of the three selected reference genes, PP2A, ASPR6 and DUSP, were used to normalise the expression of CINNAMYL ALCOHOL DEHYDROGENASE (CAD) genes across the same tissues.
Conclusions: This approach readily demonstrated the suitably of the three selected reference genes for the accurate and reliable normalisation of S. viridis RT-qPCR expression data. Further, the work reported here forms a highly useful platform for future gene expression quantification in S. viridis and can also be potentially directly translatable to other closely related and agronomically important C-4 crop species.
Details
- Title
- Reference gene identification for reliable normalisation of quantitative RT-PCR data in Setaria viridis
- Authors
- Duc Quan Nguyen (Author) - University of Newcastle AustraliaAndrew Eamens (Author) - University of Newcastle AustraliaChristopher P L Grof (Author) - University of Newcastle Australia
- Publication details
- Plant Methods, Vol.14(1), pp.1-12
- Publisher
- BioMed Central Ltd.
- Date published
- 2018
- DOI
- 10.1186/s13007-018-0293-8
- ISSN
- 1746-4811
- PMID
- 29581727; PMC5861610
- Copyright note
- The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Organisation Unit
- School of Health - Biomedicine; University of the Sunshine Coast, Queensland; School of Health and Behavioural Sciences - Legacy
- Language
- English
- Record Identifier
- 99656591802621
- Output Type
- Journal article
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