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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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Cq values for 11 candidate reference genes across experimental samples.A line across the box is depicted as the median. The box indicates the 25th and 75th percentiles. Whiskers represent the maximum and minimum values.
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pone-0103900-g002: Cq values for 11 candidate reference genes across experimental samples.A line across the box is depicted as the median. The box indicates the 25th and 75th percentiles. Whiskers represent the maximum and minimum values.

Mentions: To exclude any artificial errors in real-time PCR analysis of all the 11 candidate reference genes, three technical repetitions were performed for real-time PCR using gene-specific primers in the same cDNA pool. Meanwhile non-template controls were performed in parallel with each template and primer combination. The results showed that the single PCR product was amplified by each primer combination of the 11 candidate reference genes form various cDNA templates (Figure S2). Quantification cycle (Cq) values (the number of cycles needed for the fluorescence to reach a specific threshold level of detection) were determined in order to make comparison among each PCR run. The 11 candidate reference genes showed a relatively wide range of expression level from the lowest mean Cq value in TUA (23.36) to the highest in DNAj (32.56) with the most lying between 24 and 28 across all tested samples (Figure 2).


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)

Cq values for 11 candidate reference genes across experimental samples.A line across the box is depicted as the median. The box indicates the 25th and 75th percentiles. Whiskers represent the maximum and minimum values.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0103900-g002: Cq values for 11 candidate reference genes across experimental samples.A line across the box is depicted as the median. The box indicates the 25th and 75th percentiles. Whiskers represent the maximum and minimum values.
Mentions: To exclude any artificial errors in real-time PCR analysis of all the 11 candidate reference genes, three technical repetitions were performed for real-time PCR using gene-specific primers in the same cDNA pool. Meanwhile non-template controls were performed in parallel with each template and primer combination. The results showed that the single PCR product was amplified by each primer combination of the 11 candidate reference genes form various cDNA templates (Figure S2). Quantification cycle (Cq) values (the number of cycles needed for the fluorescence to reach a specific threshold level of detection) were determined in order to make comparison among each PCR run. The 11 candidate reference genes showed a relatively wide range of expression level from the lowest mean Cq value in TUA (23.36) to the highest in DNAj (32.56) with the most lying between 24 and 28 across all tested samples (Figure 2).

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