Abstract
BackgroundQuantitative 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 C4 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.ResultsEleven 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.ConclusionsThis 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 C4 crop species.
Highlights
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
The success of gene expression quantification via the RT-qPCR approach is entirely dependant upon the identification and use of a group of suitable reference genes for expression normalisation due to; (1) there not being any ‘universal’ reference gene(s) available for use across a wide range of phenotypically distinct plant species or experimental conditions in a single species [23, 24], and; (2) the use of only a single reference gene could lead to an incorrect functional interpretation of the expression profile of a gene [18, 21]
We assessed the suitability of eleven candidate reference genes for the accurate and reliable normalisation of gene expression quantification across thirteen developmentally distinct tissues of S. viridis, including internodes 4, 5 and 6, leaves 4, 5 and 6, inflorescence stems S1, S2 and S3, and the four developmental zones of the expanding internode, internode 5
Summary
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. Green foxtail (Setaria viridis) has recently been proposed as a potential experimental model for the study of C4 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. Setaria viridis has been identified as a model species as it possesses many desirable traits, including; (i) a small sized (~ 500 Mb), sequenced and well annotated genome; (ii) short stature (20–30 cm in height) and rapid life cycle (6–9 weeks); (iii) prolific seed production (13,000 seeds/plant), and; (iv) is amendable to Agrobacterium tumefaciens-mediated transformation [3]. The lack of a stably expressed reference gene(s) for the accurate normalisation of S. viridis RT-qPCR data has been a major hurdle for gene expression studies in this species
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
