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Evaluation and validation of reference genes for qRT-PCR normalization in Frankliniella occidentalis (Thysanoptera: Thripidae).

Zheng YT, Li HB, Lu MX, Du YZ - PLoS ONE (2014)

Bottom Line: For accurate results, the normalization of data with reference genes is particularly essential.The result demonstrated that EF-1 and RPL32 displayed the most stable expression in different developmental stages; RPL32 and GAPDH showed the most stable expression at high temperatures, while 18S and EF-1 exhibited the most stable expression at low temperatures.What is more, these validated reference genes could serve as the basis for the selection of candidate reference genes in other insects.

View Article: PubMed Central - PubMed

Affiliation: School of Horticulture and Plant Protection & Institute of Applied Entomology, Yangzhou University, Yangzhou, China.

ABSTRACT
Quantitative real time PCR (qRT-PCR) has emerged as a reliable and reproducible technique for studying gene expression analysis. For accurate results, the normalization of data with reference genes is particularly essential. Once the transcriptome sequencing of Frankliniella occidentalis was completed, numerous unigenes were identified and annotated. Unfortunately, there are no studies on the stability of reference genes used in F. occidentalis. In this work, seven candidate reference genes, including actin, 18S rRNA, H3, tubulin, GAPDH, EF-1 and RPL32, were evaluated for their suitability as normalization genes under different experimental conditions using the statistical software programs BestKeeper, geNorm, Normfinder and the comparative ΔCt method. Because the rankings of the reference genes provided by each of the four programs were different, we chose a user-friendly web-based comprehensive tool RefFinder to get the final ranking. The result demonstrated that EF-1 and RPL32 displayed the most stable expression in different developmental stages; RPL32 and GAPDH showed the most stable expression at high temperatures, while 18S and EF-1 exhibited the most stable expression at low temperatures. In this study, we validated the suitable reference genes in F. occidentalis for gene expression profiling under different experimental conditions. The choice of internal standard is very important in the normalization of the target gene expression levels, thus validating and selecting the best genes will help improve the quality of gene expression data of F. occidentalis. What is more, these validated reference genes could serve as the basis for the selection of candidate reference genes in other insects.

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Optimal number of reference genes for normalization in Frankliniella occidentalis.The pairwise variation (Vn/Vn+1) was analyzed between normalization factors NFn and NFn+1 by geNorm program to determine the optimal number of reference genes. Values <0.15 indicate that additional genes are not required for the normalization of gene expression.
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pone-0111369-g003: Optimal number of reference genes for normalization in Frankliniella occidentalis.The pairwise variation (Vn/Vn+1) was analyzed between normalization factors NFn and NFn+1 by geNorm program to determine the optimal number of reference genes. Values <0.15 indicate that additional genes are not required for the normalization of gene expression.

Mentions: The stability rankings generated by the comparative ΔCt method were largely similar with the results obtained from geNorm. However, the most stable genes as ranked by BestKeeper and NormFinder analysis were different from the results generated by geNorm and the comparative ΔCt method. The geNorm and comparative ΔCt method identified actin as the least stable gene and EF-1 as the most stable gene. In contrast, BestKeeper and NormFinder identified EF-1 as the second most stable gene. According to the results of RefFinder, the stability ranking from the most to the least stable in the developmental stages was EF-1>RPL32> tubulin >18S>GAPDH>H3>actin (Fig. 2). However, GeNorm analysis revealed that all the pair-wise variation values were above the proposed 0.15 cut-off (Fig. 3). These results indicate that normalization with three stable reference genes (EF-1, RPL32, tubulin ) was required (as suggested by the geNorm manual).


Evaluation and validation of reference genes for qRT-PCR normalization in Frankliniella occidentalis (Thysanoptera: Thripidae).

Zheng YT, Li HB, Lu MX, Du YZ - PLoS ONE (2014)

Optimal number of reference genes for normalization in Frankliniella occidentalis.The pairwise variation (Vn/Vn+1) was analyzed between normalization factors NFn and NFn+1 by geNorm program to determine the optimal number of reference genes. Values <0.15 indicate that additional genes are not required for the normalization of gene expression.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4214748&req=5

pone-0111369-g003: Optimal number of reference genes for normalization in Frankliniella occidentalis.The pairwise variation (Vn/Vn+1) was analyzed between normalization factors NFn and NFn+1 by geNorm program to determine the optimal number of reference genes. Values <0.15 indicate that additional genes are not required for the normalization of gene expression.
Mentions: The stability rankings generated by the comparative ΔCt method were largely similar with the results obtained from geNorm. However, the most stable genes as ranked by BestKeeper and NormFinder analysis were different from the results generated by geNorm and the comparative ΔCt method. The geNorm and comparative ΔCt method identified actin as the least stable gene and EF-1 as the most stable gene. In contrast, BestKeeper and NormFinder identified EF-1 as the second most stable gene. According to the results of RefFinder, the stability ranking from the most to the least stable in the developmental stages was EF-1>RPL32> tubulin >18S>GAPDH>H3>actin (Fig. 2). However, GeNorm analysis revealed that all the pair-wise variation values were above the proposed 0.15 cut-off (Fig. 3). These results indicate that normalization with three stable reference genes (EF-1, RPL32, tubulin ) was required (as suggested by the geNorm manual).

Bottom Line: For accurate results, the normalization of data with reference genes is particularly essential.The result demonstrated that EF-1 and RPL32 displayed the most stable expression in different developmental stages; RPL32 and GAPDH showed the most stable expression at high temperatures, while 18S and EF-1 exhibited the most stable expression at low temperatures.What is more, these validated reference genes could serve as the basis for the selection of candidate reference genes in other insects.

View Article: PubMed Central - PubMed

Affiliation: School of Horticulture and Plant Protection & Institute of Applied Entomology, Yangzhou University, Yangzhou, China.

ABSTRACT
Quantitative real time PCR (qRT-PCR) has emerged as a reliable and reproducible technique for studying gene expression analysis. For accurate results, the normalization of data with reference genes is particularly essential. Once the transcriptome sequencing of Frankliniella occidentalis was completed, numerous unigenes were identified and annotated. Unfortunately, there are no studies on the stability of reference genes used in F. occidentalis. In this work, seven candidate reference genes, including actin, 18S rRNA, H3, tubulin, GAPDH, EF-1 and RPL32, were evaluated for their suitability as normalization genes under different experimental conditions using the statistical software programs BestKeeper, geNorm, Normfinder and the comparative ΔCt method. Because the rankings of the reference genes provided by each of the four programs were different, we chose a user-friendly web-based comprehensive tool RefFinder to get the final ranking. The result demonstrated that EF-1 and RPL32 displayed the most stable expression in different developmental stages; RPL32 and GAPDH showed the most stable expression at high temperatures, while 18S and EF-1 exhibited the most stable expression at low temperatures. In this study, we validated the suitable reference genes in F. occidentalis for gene expression profiling under different experimental conditions. The choice of internal standard is very important in the normalization of the target gene expression levels, thus validating and selecting the best genes will help improve the quality of gene expression data of F. occidentalis. What is more, these validated reference genes could serve as the basis for the selection of candidate reference genes in other insects.

Show MeSH