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Selection of reference genes for quantitative gene expression studies in Platycladus orientalis (Cupressaceae) Using real-time PCR.

Chang E, Shi S, Liu J, Cheng T, Xue L, Yang X, Yang W, Lan Q, Jiang Z - PLoS ONE (2012)

Bottom Line: In parallel, aTUB and UBC were used singly or in combination as reference genes to examine the expression levels of NAC (a homolog of AtNAC2) in plants subjected to various treatments with qRT-PCR.The results further proved the reliability of the two selected reference genes.Our study will benefit future research on the expression of genes in response to stress/senescence in P. orientalis and other members of the Cupressaceae.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Tree Genetics and Breeding, Research Institute of Forestry, Chinese Academy of Forestry, Beijing, People's Republic of China.

ABSTRACT
Platycladus orientalis is a tree species that is highly resistant, widely adaptable, and long-lived, with lifespans of even thousands of years. To explore the mechanisms underlying these characteristics, gene expressions have been investigated at the transcriptome level by RNA-seq combined with a digital gene expression (DGE) technique. So, it is crucial to have a reliable set of reference genes to normalize the expressions of genes in P. orientalis under various conditions using the most accurate and sensitive method of quantitative real-time PCR (qRT-PCR). In this study, we selected 10 reference gene candidates from transcriptome data of P. orientalis, and examined their expression profiles by qRT-PCR using 29 different samples of P. orientalis, which were collected from plants of different ages, different tissues, and plants subjected to different treatments including cold, heat, salinity, polyethylene glycol (PEG), and abscisic acid (ABA). Three analytical software packages (geNorm, Bestkeeper, and NormFinder) were used to assess the stability of gene expression. The results showed that ubiquitin-conjugating enzyme E2 (UBC) and alpha-tubulin (aTUB) were the optimum pair of reference genes at all developmental stages and under all stress conditions. ACT7 was the most stable gene across different tissues and cold-treated samples, while UBQ was the most stably expressed reference gene for NaCl- and ABA-treated samples. In parallel, aTUB and UBC were used singly or in combination as reference genes to examine the expression levels of NAC (a homolog of AtNAC2) in plants subjected to various treatments with qRT-PCR. The results further proved the reliability of the two selected reference genes. Our study will benefit future research on the expression of genes in response to stress/senescence in P. orientalis and other members of the Cupressaceae.

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Expression profiles of NAC in different-aged tissues and in response to stresses in Platycladus orientalis (as determined by qRT-PCR with UBC and aTUB in combination as reference genes).
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pone-0033278-g004: Expression profiles of NAC in different-aged tissues and in response to stresses in Platycladus orientalis (as determined by qRT-PCR with UBC and aTUB in combination as reference genes).

Mentions: To further evaluate the reliability of the top two reference genes aTUB and UBC, NAC transcription factor AtNAC2 (At5g39610; also named ANAC092), which plays a crucial role in protecting plants against abiotic stresses, as well as in leaf senescence [45], was selected to qRT-PCR analysis with aTUB and UBC singly or in combination as reference genes. In our previous study, we found that the NAC domain gene from P. orientalis, a homolog of AtNAC2 in A. thaliana, was differentially expressed between young and old trees by using RNA-seq combined with a DGE technique (data not shown). Therefore, this gene was selected to further evaluate the reliability of the reference genes by qRT-PCR. We selected aTUB and UBC in combination as the reference genes and determined their average expression patterns (Fig. 4). The highest level of NAC expression was in leaves of 5-year-old P. orientalis. The expression of NAC in leaves increased significantly from 20- to 2000-year-old individuals of P. orientalis (P < 0.05) (Fig. 4A), which matches the result obtained from the DGE analysis. NAC showed evidently different expression levels in the tissues, such as seeds, fruits, roots, stems, and leaves (P < 0.05) (Fig. 4B). Cold and heat stress significantly increased the expression of NAC (Fig. 4C) by 8.0 and 5.7 folds, respectively, at 48 h compared with that at 0 h (P < 0.05). The expression profiles of NAC showed similar trends under NaCl, PEG, and ABA treatments, and its abundance increased to maximum levels at 24 h, increasing to 12.9, 5.6 and 12.2 folds compared with that at 0 h, respectively (P < 0.05) (Fig. 4C). In addition, the expression patterns of NAC showed similar trends to those of aTUB and UBC as internal controls either singly or in combination (Fig. 4; Fig. S1). There were no significant differences in the expression patterns of NAC using either aTUB or UBC singly as the internal control (P > 0.05), although it seems that there was a bit of difference between aTUB and UBC at the 48-hour treatments (P  =  0.059) (Fig. S1), which further indicated that aTUB and UBC were suitable as reference genes.


Selection of reference genes for quantitative gene expression studies in Platycladus orientalis (Cupressaceae) Using real-time PCR.

Chang E, Shi S, Liu J, Cheng T, Xue L, Yang X, Yang W, Lan Q, Jiang Z - PLoS ONE (2012)

Expression profiles of NAC in different-aged tissues and in response to stresses in Platycladus orientalis (as determined by qRT-PCR with UBC and aTUB in combination as reference genes).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3316566&req=5

pone-0033278-g004: Expression profiles of NAC in different-aged tissues and in response to stresses in Platycladus orientalis (as determined by qRT-PCR with UBC and aTUB in combination as reference genes).
Mentions: To further evaluate the reliability of the top two reference genes aTUB and UBC, NAC transcription factor AtNAC2 (At5g39610; also named ANAC092), which plays a crucial role in protecting plants against abiotic stresses, as well as in leaf senescence [45], was selected to qRT-PCR analysis with aTUB and UBC singly or in combination as reference genes. In our previous study, we found that the NAC domain gene from P. orientalis, a homolog of AtNAC2 in A. thaliana, was differentially expressed between young and old trees by using RNA-seq combined with a DGE technique (data not shown). Therefore, this gene was selected to further evaluate the reliability of the reference genes by qRT-PCR. We selected aTUB and UBC in combination as the reference genes and determined their average expression patterns (Fig. 4). The highest level of NAC expression was in leaves of 5-year-old P. orientalis. The expression of NAC in leaves increased significantly from 20- to 2000-year-old individuals of P. orientalis (P < 0.05) (Fig. 4A), which matches the result obtained from the DGE analysis. NAC showed evidently different expression levels in the tissues, such as seeds, fruits, roots, stems, and leaves (P < 0.05) (Fig. 4B). Cold and heat stress significantly increased the expression of NAC (Fig. 4C) by 8.0 and 5.7 folds, respectively, at 48 h compared with that at 0 h (P < 0.05). The expression profiles of NAC showed similar trends under NaCl, PEG, and ABA treatments, and its abundance increased to maximum levels at 24 h, increasing to 12.9, 5.6 and 12.2 folds compared with that at 0 h, respectively (P < 0.05) (Fig. 4C). In addition, the expression patterns of NAC showed similar trends to those of aTUB and UBC as internal controls either singly or in combination (Fig. 4; Fig. S1). There were no significant differences in the expression patterns of NAC using either aTUB or UBC singly as the internal control (P > 0.05), although it seems that there was a bit of difference between aTUB and UBC at the 48-hour treatments (P  =  0.059) (Fig. S1), which further indicated that aTUB and UBC were suitable as reference genes.

Bottom Line: In parallel, aTUB and UBC were used singly or in combination as reference genes to examine the expression levels of NAC (a homolog of AtNAC2) in plants subjected to various treatments with qRT-PCR.The results further proved the reliability of the two selected reference genes.Our study will benefit future research on the expression of genes in response to stress/senescence in P. orientalis and other members of the Cupressaceae.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Tree Genetics and Breeding, Research Institute of Forestry, Chinese Academy of Forestry, Beijing, People's Republic of China.

ABSTRACT
Platycladus orientalis is a tree species that is highly resistant, widely adaptable, and long-lived, with lifespans of even thousands of years. To explore the mechanisms underlying these characteristics, gene expressions have been investigated at the transcriptome level by RNA-seq combined with a digital gene expression (DGE) technique. So, it is crucial to have a reliable set of reference genes to normalize the expressions of genes in P. orientalis under various conditions using the most accurate and sensitive method of quantitative real-time PCR (qRT-PCR). In this study, we selected 10 reference gene candidates from transcriptome data of P. orientalis, and examined their expression profiles by qRT-PCR using 29 different samples of P. orientalis, which were collected from plants of different ages, different tissues, and plants subjected to different treatments including cold, heat, salinity, polyethylene glycol (PEG), and abscisic acid (ABA). Three analytical software packages (geNorm, Bestkeeper, and NormFinder) were used to assess the stability of gene expression. The results showed that ubiquitin-conjugating enzyme E2 (UBC) and alpha-tubulin (aTUB) were the optimum pair of reference genes at all developmental stages and under all stress conditions. ACT7 was the most stable gene across different tissues and cold-treated samples, while UBQ was the most stably expressed reference gene for NaCl- and ABA-treated samples. In parallel, aTUB and UBC were used singly or in combination as reference genes to examine the expression levels of NAC (a homolog of AtNAC2) in plants subjected to various treatments with qRT-PCR. The results further proved the reliability of the two selected reference genes. Our study will benefit future research on the expression of genes in response to stress/senescence in P. orientalis and other members of the Cupressaceae.

Show MeSH
Related in: MedlinePlus