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Selection of reference genes for gene expression studies in Siberian Apricot (Prunus sibirica L.) Germplasm using quantitative real-time PCR.

Niu J, Zhu B, Cai J, Li P, Wang L, Dai H, Qiu L, Yu H, Ha D, Zhao H, Zhang Z, Lin S - PLoS ONE (2014)

Bottom Line: Moreover, UBC was selected as the most stably expressed gene by both algorithms in different Siberian Apricot seed samples.We also detected that a set of three genes (ACT, CYP and UBC) by geNorm as control for normalization could lead to accurate results.These obtained experimental results could make an important contribution to normalize real-time PCR data for gene expression analysis in Siberian Apricot Germplasm.

View Article: PubMed Central - PubMed

Affiliation: College of Biological Sciences and Biotechnology, College of Nature Conservation, National Engineering Laboratory for Tree Breeding, Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants, Ministry of Education, Beijing Forestry University, Beijing, China.

ABSTRACT
Quantitative real time reverse transcription polymerase chain reaction has been applied in a vast range of studies of gene expression analysis. However, real-time PCR data must be normalized with one or more reference genes. In this study, eleven putative consistently expressed genes (ACT, TUA, TUB, CYP, DNAj, ELFA, F-box27, RPL12, GAPDH, UBC and UBQ) in nine Siberian Apricot Germplasms (including much variability) were evaluated for their potential as references for the normalization of gene expression by NormFinder and geNorm programs. From our studies, ACT, UBC, CYP, UBQ and RPL12 as suitable for normalization were identified by geNorm, while UBC and CYP as the best pair by NormFinder. Moreover, UBC was selected as the most stably expressed gene by both algorithms in different Siberian Apricot seed samples. We also detected that a set of three genes (ACT, CYP and UBC) by geNorm as control for normalization could lead to accurate results. Furthermore, the expression levels of oleosin gene were analyzed to validate the suitability of the selected reference genes. These obtained experimental results could make an important contribution to normalize real-time PCR data for gene expression analysis in Siberian Apricot Germplasm.

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Expression profiles of oleosin gene in different Siberian Apricot Germplasms.Expression ratios of oleosin for the experimental calculated using (1) CYP and UBC for NormFinder; (2) CYP, UBC and ACT for geNorm; (3) ACT, UBC, CYP, UBQ and RPL12 for geNorm; (4) the stable reference gene UBC; (5) the unsatisfactory reference gene F-box27.
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pone-0103900-g006: Expression profiles of oleosin gene in different Siberian Apricot Germplasms.Expression ratios of oleosin for the experimental calculated using (1) CYP and UBC for NormFinder; (2) CYP, UBC and ACT for geNorm; (3) ACT, UBC, CYP, UBQ and RPL12 for geNorm; (4) the stable reference gene UBC; (5) the unsatisfactory reference gene F-box27.

Mentions: The expression level of the oleosin gene was performed as an example to show the effect of the choice of reference genes on the expression profiling of other genes. The oleosin expression was analyzed in different Siberian Apricot Germplasms by using the various combinations of top reference genes indicated by each of the three methods: UBC, CYP and ACT, and CYP, UBQ, ACT, RPL12 and UBC as indicated by geNorm; CYP and UBC as indicated by NormFinder. In addition, we also used the optimal (UBC) and the inadequate (F-box27) genes as reference genes. As shown in Figure 6, normalization using the unstable reference F-box27 gene led to erroneous estimation of the target gene. When the UBC was used as a unique reference, the expressions of oleosin in Tianjing and Yanji were significantly overestimated. The results from geNorm (three and five genes) and NormFinder had a better parallel veracity.


Selection of reference genes for gene expression studies in Siberian Apricot (Prunus sibirica L.) Germplasm using quantitative real-time PCR.

Niu J, Zhu B, Cai J, Li P, Wang L, Dai H, Qiu L, Yu H, Ha D, Zhao H, Zhang Z, Lin S - PLoS ONE (2014)

Expression profiles of oleosin gene in different Siberian Apricot Germplasms.Expression ratios of oleosin for the experimental calculated using (1) CYP and UBC for NormFinder; (2) CYP, UBC and ACT for geNorm; (3) ACT, UBC, CYP, UBQ and RPL12 for geNorm; (4) the stable reference gene UBC; (5) the unsatisfactory reference gene F-box27.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126684&req=5

pone-0103900-g006: Expression profiles of oleosin gene in different Siberian Apricot Germplasms.Expression ratios of oleosin for the experimental calculated using (1) CYP and UBC for NormFinder; (2) CYP, UBC and ACT for geNorm; (3) ACT, UBC, CYP, UBQ and RPL12 for geNorm; (4) the stable reference gene UBC; (5) the unsatisfactory reference gene F-box27.
Mentions: The expression level of the oleosin gene was performed as an example to show the effect of the choice of reference genes on the expression profiling of other genes. The oleosin expression was analyzed in different Siberian Apricot Germplasms by using the various combinations of top reference genes indicated by each of the three methods: UBC, CYP and ACT, and CYP, UBQ, ACT, RPL12 and UBC as indicated by geNorm; CYP and UBC as indicated by NormFinder. In addition, we also used the optimal (UBC) and the inadequate (F-box27) genes as reference genes. As shown in Figure 6, normalization using the unstable reference F-box27 gene led to erroneous estimation of the target gene. When the UBC was used as a unique reference, the expressions of oleosin in Tianjing and Yanji were significantly overestimated. The results from geNorm (three and five genes) and NormFinder had a better parallel veracity.

Bottom Line: Moreover, UBC was selected as the most stably expressed gene by both algorithms in different Siberian Apricot seed samples.We also detected that a set of three genes (ACT, CYP and UBC) by geNorm as control for normalization could lead to accurate results.These obtained experimental results could make an important contribution to normalize real-time PCR data for gene expression analysis in Siberian Apricot Germplasm.

View Article: PubMed Central - PubMed

Affiliation: College of Biological Sciences and Biotechnology, College of Nature Conservation, National Engineering Laboratory for Tree Breeding, Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants, Ministry of Education, Beijing Forestry University, Beijing, China.

ABSTRACT
Quantitative real time reverse transcription polymerase chain reaction has been applied in a vast range of studies of gene expression analysis. However, real-time PCR data must be normalized with one or more reference genes. In this study, eleven putative consistently expressed genes (ACT, TUA, TUB, CYP, DNAj, ELFA, F-box27, RPL12, GAPDH, UBC and UBQ) in nine Siberian Apricot Germplasms (including much variability) were evaluated for their potential as references for the normalization of gene expression by NormFinder and geNorm programs. From our studies, ACT, UBC, CYP, UBQ and RPL12 as suitable for normalization were identified by geNorm, while UBC and CYP as the best pair by NormFinder. Moreover, UBC was selected as the most stably expressed gene by both algorithms in different Siberian Apricot seed samples. We also detected that a set of three genes (ACT, CYP and UBC) by geNorm as control for normalization could lead to accurate results. Furthermore, the expression levels of oleosin gene were analyzed to validate the suitability of the selected reference genes. These obtained experimental results could make an important contribution to normalize real-time PCR data for gene expression analysis in Siberian Apricot Germplasm.

Show MeSH