Limits...
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.

Show MeSH
Pairwise variation analysis of normalization factors to determine the optimal number of reference genes.The cut-off value of 0.15, below which the inclusion of an additional reference gene is not required, is indicated by a discontinuous line.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4126684&req=5

pone-0103900-g004: Pairwise variation analysis of normalization factors to determine the optimal number of reference genes.The cut-off value of 0.15, below which the inclusion of an additional reference gene is not required, is indicated by a discontinuous line.

Mentions: The expression stability value of the 11 candidate reference genes was calculated by the geNorm program and then the reference genes TUB and RPL12 were identified as the two most stably-expressed genes (Figure 3A), according to their M values defined as the average pairwise variation of that gene relative to all other potential reference genes in a set of cDNA samples. Since two pairs of reference genes (TUB and TUA; UBQ and UBC) were suspected to be coregulated, we removed both TUB and UBQ from analysis. The occurrence of a shift in ranking after the removal of those suspected coregulated genes (TUB and UBQ) showed that ACT and UBC were the best pair (Figure 3B). To determine whether the two pairs of reference genes were coregulated genes, TUB and UBQ were independently removed (Figure 3C and Figure 3D). The resulting ranking indicated that TUB and TUA were coregulated. In general, selecting more than two reference genes as an internal control can help significantly with the correction experimental error, providing get more reliable results. This is critical for accurate quantification of the genes, especially small expression differences in gene expression studies [20]. In order to select the ideal number of reference genes required for accurate normalization, the pairwise variation (Vn/Vn+1) was calculated by the geNorm algorithm. Vn values were calculated by stepwise inclusion of more reference genes until the (n+1) gene made no significant contribution to the newly calculated normalization factor [21]. According to the analysis, a value of 0.15 is usually regarded as the selection threshold value, for example V2/3<0.15 means that 2 reference genes could be used for normalization [10]. In our present study, V5/6 (0.148)<0.15<V4/5 (0.151) suggested that five reference genes were the best option for accurate normalization (Figure 4). However, we found the value of V3/4 (0.151) to be very close to 0.15, and three reference genes were used for attempted normalization. Finally, UBC, ACT, CYP, UBQ and RPL12 were identified as the most suitable reference genes, and UBC, CYP and ACT were tentatively selected for normalization by geNorm.


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)

Pairwise variation analysis of normalization factors to determine the optimal number of reference genes.The cut-off value of 0.15, below which the inclusion of an additional reference gene is not required, is indicated by a discontinuous line.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0103900-g004: Pairwise variation analysis of normalization factors to determine the optimal number of reference genes.The cut-off value of 0.15, below which the inclusion of an additional reference gene is not required, is indicated by a discontinuous line.
Mentions: The expression stability value of the 11 candidate reference genes was calculated by the geNorm program and then the reference genes TUB and RPL12 were identified as the two most stably-expressed genes (Figure 3A), according to their M values defined as the average pairwise variation of that gene relative to all other potential reference genes in a set of cDNA samples. Since two pairs of reference genes (TUB and TUA; UBQ and UBC) were suspected to be coregulated, we removed both TUB and UBQ from analysis. The occurrence of a shift in ranking after the removal of those suspected coregulated genes (TUB and UBQ) showed that ACT and UBC were the best pair (Figure 3B). To determine whether the two pairs of reference genes were coregulated genes, TUB and UBQ were independently removed (Figure 3C and Figure 3D). The resulting ranking indicated that TUB and TUA were coregulated. In general, selecting more than two reference genes as an internal control can help significantly with the correction experimental error, providing get more reliable results. This is critical for accurate quantification of the genes, especially small expression differences in gene expression studies [20]. In order to select the ideal number of reference genes required for accurate normalization, the pairwise variation (Vn/Vn+1) was calculated by the geNorm algorithm. Vn values were calculated by stepwise inclusion of more reference genes until the (n+1) gene made no significant contribution to the newly calculated normalization factor [21]. According to the analysis, a value of 0.15 is usually regarded as the selection threshold value, for example V2/3<0.15 means that 2 reference genes could be used for normalization [10]. In our present study, V5/6 (0.148)<0.15<V4/5 (0.151) suggested that five reference genes were the best option for accurate normalization (Figure 4). However, we found the value of V3/4 (0.151) to be very close to 0.15, and three reference genes were used for attempted normalization. Finally, UBC, ACT, CYP, UBQ and RPL12 were identified as the most suitable reference genes, and UBC, CYP and ACT were tentatively selected for normalization by geNorm.

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