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Validation of Reference Genes for Accurate Normalization of Gene Expression in Lilium davidii var. unicolor for Real Time Quantitative PCR.

Li X, Cheng J, Zhang J, Teixeira da Silva JA, Wang C, Sun H - PLoS ONE (2015)

Bottom Line: The requirement of suitable reference genes for normalization has become increasingly significant and exigent.In leaves, scales and roots under stress treatments, FP, ACT and AP4, respectively showed the most stable expression.This study provides a guide for the selection of a reference gene under different experimental conditions, and will benefit future research on more accurate gene expression studies in a wide variety of Lilium genotypes.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Protected Horticulture of Education Ministry and Liaoning Province, College of Horticulture, Shenyang Agricultural University, Shenyang, P R China.

ABSTRACT
Lilium is an important commercial market flower bulb. qRT-PCR is an extremely important technique to track gene expression levels. The requirement of suitable reference genes for normalization has become increasingly significant and exigent. The expression of internal control genes in living organisms varies considerably under different experimental conditions. For economically important Lilium, only a limited number of reference genes applied in qRT-PCR have been reported to date. In this study, the expression stability of 12 candidate genes including α-TUB, β-TUB, ACT, eIF, GAPDH, UBQ, UBC, 18S, 60S, AP4, FP, and RH2, in a diverse set of 29 samples representing different developmental processes, three stress treatments (cold, heat, and salt) and different organs, has been evaluated. For different organs, the combination of ACT, GAPDH, and UBQ is appropriate whereas ACT together with AP4, or ACT along with GAPDH is suitable for normalization of leaves and scales at different developmental stages, respectively. In leaves, scales and roots under stress treatments, FP, ACT and AP4, respectively showed the most stable expression. This study provides a guide for the selection of a reference gene under different experimental conditions, and will benefit future research on more accurate gene expression studies in a wide variety of Lilium genotypes.

No MeSH data available.


Related in: MedlinePlus

Amplification of the candidate reference genes from cDNA templates.Agarose gel electrophoresis showing amplification of a specific PCR product of the expect size for each gene.
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pone.0141323.g001: Amplification of the candidate reference genes from cDNA templates.Agarose gel electrophoresis showing amplification of a specific PCR product of the expect size for each gene.

Mentions: In order to determine the specificity and efficiency of primers, 2% agarose gel electrophoresis analyses were performed to check the amplicons of the candidate reference genes derived from all templates. All the primers pairs amplified single fragments of the expected size (Fig 1). Sequencing analyses showed that all genes were 100% identical to their original genes deposited in the GenBank database (unpublished data); the sequences data of these genes are shown in S1 File. In addition, the specificity of the amplicons was confirmed by the presence of a single peak in melting curve analyses following qRT-PCR (S1 Fig), and no products were detected in negative controls. A standard curve was generated using 10-fold serial dilutions of pooled cDNA and the slopes of standard curves were used to check R2 values and PCR efficiency. The PCR efficiencies ranged from 95% to 105%, which are well within the acceptable range of 90–105% of qRT-PCR and suitable for further gene expression analysis by qRT-PCR (Table 1). In addition, the standard curves showed good linear relationships (R2 values ranged from 0.9910 to 0.9998) between Ct and the log-transformed copy numbers.


Validation of Reference Genes for Accurate Normalization of Gene Expression in Lilium davidii var. unicolor for Real Time Quantitative PCR.

Li X, Cheng J, Zhang J, Teixeira da Silva JA, Wang C, Sun H - PLoS ONE (2015)

Amplification of the candidate reference genes from cDNA templates.Agarose gel electrophoresis showing amplification of a specific PCR product of the expect size for each gene.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4624937&req=5

pone.0141323.g001: Amplification of the candidate reference genes from cDNA templates.Agarose gel electrophoresis showing amplification of a specific PCR product of the expect size for each gene.
Mentions: In order to determine the specificity and efficiency of primers, 2% agarose gel electrophoresis analyses were performed to check the amplicons of the candidate reference genes derived from all templates. All the primers pairs amplified single fragments of the expected size (Fig 1). Sequencing analyses showed that all genes were 100% identical to their original genes deposited in the GenBank database (unpublished data); the sequences data of these genes are shown in S1 File. In addition, the specificity of the amplicons was confirmed by the presence of a single peak in melting curve analyses following qRT-PCR (S1 Fig), and no products were detected in negative controls. A standard curve was generated using 10-fold serial dilutions of pooled cDNA and the slopes of standard curves were used to check R2 values and PCR efficiency. The PCR efficiencies ranged from 95% to 105%, which are well within the acceptable range of 90–105% of qRT-PCR and suitable for further gene expression analysis by qRT-PCR (Table 1). In addition, the standard curves showed good linear relationships (R2 values ranged from 0.9910 to 0.9998) between Ct and the log-transformed copy numbers.

Bottom Line: The requirement of suitable reference genes for normalization has become increasingly significant and exigent.In leaves, scales and roots under stress treatments, FP, ACT and AP4, respectively showed the most stable expression.This study provides a guide for the selection of a reference gene under different experimental conditions, and will benefit future research on more accurate gene expression studies in a wide variety of Lilium genotypes.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Protected Horticulture of Education Ministry and Liaoning Province, College of Horticulture, Shenyang Agricultural University, Shenyang, P R China.

ABSTRACT
Lilium is an important commercial market flower bulb. qRT-PCR is an extremely important technique to track gene expression levels. The requirement of suitable reference genes for normalization has become increasingly significant and exigent. The expression of internal control genes in living organisms varies considerably under different experimental conditions. For economically important Lilium, only a limited number of reference genes applied in qRT-PCR have been reported to date. In this study, the expression stability of 12 candidate genes including α-TUB, β-TUB, ACT, eIF, GAPDH, UBQ, UBC, 18S, 60S, AP4, FP, and RH2, in a diverse set of 29 samples representing different developmental processes, three stress treatments (cold, heat, and salt) and different organs, has been evaluated. For different organs, the combination of ACT, GAPDH, and UBQ is appropriate whereas ACT together with AP4, or ACT along with GAPDH is suitable for normalization of leaves and scales at different developmental stages, respectively. In leaves, scales and roots under stress treatments, FP, ACT and AP4, respectively showed the most stable expression. This study provides a guide for the selection of a reference gene under different experimental conditions, and will benefit future research on more accurate gene expression studies in a wide variety of Lilium genotypes.

No MeSH data available.


Related in: MedlinePlus