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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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Performance of the amplification primers.Amplicons obtained by real-time PCR using cDNA (up) and gDNA (down) as template and electrophoresis using agarose gel (1.5%). The amplification primers from left to right are ACT, TUA, TUB, CYP, DNAj, ELFA, F-box27, RPL12, GAPDH, UBC and UBQ.
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pone-0103900-g001: Performance of the amplification primers.Amplicons obtained by real-time PCR using cDNA (up) and gDNA (down) as template and electrophoresis using agarose gel (1.5%). The amplification primers from left to right are ACT, TUA, TUB, CYP, DNAj, ELFA, F-box27, RPL12, GAPDH, UBC and UBQ.

Mentions: Agarose gel electrophoresis and melting curve analyses were performed following the RT-qPCR experiment, to determine specificity of primers designed in the current study. The primer pairs all amplified the expected size of the single PCR product (Figure 1). In addition, the specificity of amplicon was confirmed by the presence of a single peak during the melt curve and sequencing analysis (Figure S2). Average amplification efficiency varied from 99.2% and 105.9% (Table 3).


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)

Performance of the amplification primers.Amplicons obtained by real-time PCR using cDNA (up) and gDNA (down) as template and electrophoresis using agarose gel (1.5%). The amplification primers from left to right are ACT, TUA, TUB, CYP, DNAj, ELFA, F-box27, RPL12, GAPDH, UBC and UBQ.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0103900-g001: Performance of the amplification primers.Amplicons obtained by real-time PCR using cDNA (up) and gDNA (down) as template and electrophoresis using agarose gel (1.5%). The amplification primers from left to right are ACT, TUA, TUB, CYP, DNAj, ELFA, F-box27, RPL12, GAPDH, UBC and UBQ.
Mentions: Agarose gel electrophoresis and melting curve analyses were performed following the RT-qPCR experiment, to determine specificity of primers designed in the current study. The primer pairs all amplified the expected size of the single PCR product (Figure 1). In addition, the specificity of amplicon was confirmed by the presence of a single peak during the melt curve and sequencing analysis (Figure S2). Average amplification efficiency varied from 99.2% and 105.9% (Table 3).

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