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Validation of reference genes in Solenopsis invicta in different developmental stages, castes and tissues.

Cheng D, Zhang Z, He X, Liang G - PLoS ONE (2013)

Bottom Line: Furthermore, the optimal number of reference gene(s) was determined by the pairwise variation value.Our data showed that two of the five candidate genes, rpl18 and ef1-beta, were the most suitable reference genes because they have the most stable expression among different developmental stages, castes and tissues in S. invicta.Although widely used as reference gene in other species, in S. invicta the act gene has high variation in expression and was consequently excluded as a reliable reference gene.

View Article: PubMed Central - PubMed

Affiliation: Department of Entomology, South China Agricultural University, Guangzhou, Guangdong, People's Republic China.

ABSTRACT
To accurately assess gene expression levels, it is essential to normalize real-time quantitative PCR (RT-qPCR) data with suitable internal reference genes. For the red imported fire ant, Solenopsis invicta, reliable reference genes to assess the transcript expression levels of the target genes have not been previously investigated. In this study, we examined the expression levels of five candidate reference genes (rpl18, ef1-beta, act, GAPDH, and tbp) in different developmental stages, castes and tissues of S. invicta. To evaluate the suitability of these genes as endogenous controls, three software-based approaches (geNorm, BestKeeper and NormFinder) and one web-based comprehensive tool (RefFinder) were used to analyze and rank the tested genes. Furthermore, the optimal number of reference gene(s) was determined by the pairwise variation value. Our data showed that two of the five candidate genes, rpl18 and ef1-beta, were the most suitable reference genes because they have the most stable expression among different developmental stages, castes and tissues in S. invicta. Although widely used as reference gene in other species, in S. invicta the act gene has high variation in expression and was consequently excluded as a reliable reference gene. The two validated reference genes, rpl18 and ef1-beta, can be widely used for quantification of target gene expression with RT-qPCR technology in S. invicta.

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Threshold cycles (Ct) comparison of five reference genes.a: Developmental stages; b: Castes; c: Tissues. P values are given for each of the five putative genes (One-Way ANOVA). Developmental stages: Larvae (L), Pupae (P), Adult (A); Castes: Worker (W), Alate Male (M), Alate Female (F); Tissues: Antenna (An), Head without antennae (He), Thorax (Th), Leg (Pr) and Abdomen (Ab).
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pone-0057718-g001: Threshold cycles (Ct) comparison of five reference genes.a: Developmental stages; b: Castes; c: Tissues. P values are given for each of the five putative genes (One-Way ANOVA). Developmental stages: Larvae (L), Pupae (P), Adult (A); Castes: Worker (W), Alate Male (M), Alate Female (F); Tissues: Antenna (An), Head without antennae (He), Thorax (Th), Leg (Pr) and Abdomen (Ab).

Mentions: The expression profiles of the five candidate reference genes were assessed in different developmental stages, castes and tissues (Figure 1a, 1b, 1c). The distributions of the Ct values for each gene in samples from different developmental stages were not significantly different, with probability values (P) greater than 0.068 (One-Way ANOVA, Figure 1a). Two of five candidate genes, rpl18 and ef1-beta had no significantly different Ct values between castes, while other three candidate genes, act, tbp and GAPDH had significantly different Ct values between castes with P≤0.018 (One-Way ANOVA, Figure 1b). The Ct values for rpl18, ef1-beta, act and GAPDH were significantly different among different tissues (One-Way ANOVA, P≤0.021), whereas no significant differences were detected in tbp expression in different tissues (Figure 1c).


Validation of reference genes in Solenopsis invicta in different developmental stages, castes and tissues.

Cheng D, Zhang Z, He X, Liang G - PLoS ONE (2013)

Threshold cycles (Ct) comparison of five reference genes.a: Developmental stages; b: Castes; c: Tissues. P values are given for each of the five putative genes (One-Way ANOVA). Developmental stages: Larvae (L), Pupae (P), Adult (A); Castes: Worker (W), Alate Male (M), Alate Female (F); Tissues: Antenna (An), Head without antennae (He), Thorax (Th), Leg (Pr) and Abdomen (Ab).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585193&req=5

pone-0057718-g001: Threshold cycles (Ct) comparison of five reference genes.a: Developmental stages; b: Castes; c: Tissues. P values are given for each of the five putative genes (One-Way ANOVA). Developmental stages: Larvae (L), Pupae (P), Adult (A); Castes: Worker (W), Alate Male (M), Alate Female (F); Tissues: Antenna (An), Head without antennae (He), Thorax (Th), Leg (Pr) and Abdomen (Ab).
Mentions: The expression profiles of the five candidate reference genes were assessed in different developmental stages, castes and tissues (Figure 1a, 1b, 1c). The distributions of the Ct values for each gene in samples from different developmental stages were not significantly different, with probability values (P) greater than 0.068 (One-Way ANOVA, Figure 1a). Two of five candidate genes, rpl18 and ef1-beta had no significantly different Ct values between castes, while other three candidate genes, act, tbp and GAPDH had significantly different Ct values between castes with P≤0.018 (One-Way ANOVA, Figure 1b). The Ct values for rpl18, ef1-beta, act and GAPDH were significantly different among different tissues (One-Way ANOVA, P≤0.021), whereas no significant differences were detected in tbp expression in different tissues (Figure 1c).

Bottom Line: Furthermore, the optimal number of reference gene(s) was determined by the pairwise variation value.Our data showed that two of the five candidate genes, rpl18 and ef1-beta, were the most suitable reference genes because they have the most stable expression among different developmental stages, castes and tissues in S. invicta.Although widely used as reference gene in other species, in S. invicta the act gene has high variation in expression and was consequently excluded as a reliable reference gene.

View Article: PubMed Central - PubMed

Affiliation: Department of Entomology, South China Agricultural University, Guangzhou, Guangdong, People's Republic China.

ABSTRACT
To accurately assess gene expression levels, it is essential to normalize real-time quantitative PCR (RT-qPCR) data with suitable internal reference genes. For the red imported fire ant, Solenopsis invicta, reliable reference genes to assess the transcript expression levels of the target genes have not been previously investigated. In this study, we examined the expression levels of five candidate reference genes (rpl18, ef1-beta, act, GAPDH, and tbp) in different developmental stages, castes and tissues of S. invicta. To evaluate the suitability of these genes as endogenous controls, three software-based approaches (geNorm, BestKeeper and NormFinder) and one web-based comprehensive tool (RefFinder) were used to analyze and rank the tested genes. Furthermore, the optimal number of reference gene(s) was determined by the pairwise variation value. Our data showed that two of the five candidate genes, rpl18 and ef1-beta, were the most suitable reference genes because they have the most stable expression among different developmental stages, castes and tissues in S. invicta. Although widely used as reference gene in other species, in S. invicta the act gene has high variation in expression and was consequently excluded as a reliable reference gene. The two validated reference genes, rpl18 and ef1-beta, can be widely used for quantification of target gene expression with RT-qPCR technology in S. invicta.

Show MeSH