Limits...
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.

Show MeSH
The optimal number of reference genes for normalization by geNorm analysis.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585193&req=5

pone-0057718-g002: The optimal number of reference genes for normalization by geNorm analysis.

Mentions: Pairwise variation values were determined individually for the developmental stages, castes and tissues datasets. Results showed that pairwise variation values for V2/3 were below the cut-off value of 0.15 in all scenarios (Figure 2). Thus, no third control gene was required when RT-qPCR was carried out in any of the three treatments, meaning that the use of two most stable candidate genes (i.e., rpl18 and ef1-beta) as reference genes would be sufficient for a stable and valid reference in RT-qPCR analysis.


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)

The optimal number of reference genes for normalization by geNorm analysis.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585193&req=5

pone-0057718-g002: The optimal number of reference genes for normalization by geNorm analysis.
Mentions: Pairwise variation values were determined individually for the developmental stages, castes and tissues datasets. Results showed that pairwise variation values for V2/3 were below the cut-off value of 0.15 in all scenarios (Figure 2). Thus, no third control gene was required when RT-qPCR was carried out in any of the three treatments, meaning that the use of two most stable candidate genes (i.e., rpl18 and ef1-beta) as reference genes would be sufficient for a stable and valid reference in RT-qPCR analysis.

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