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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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Validation of reference gene selection.A) Relative expression levels of Hop in different developmental stages; B) Exposed to high temperatures; C) Exposed to low temperatures (P<0.05, Tukey s-b(k); n = 4).
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pone-0111369-g004: Validation of reference gene selection.A) Relative expression levels of Hop in different developmental stages; B) Exposed to high temperatures; C) Exposed to low temperatures (P<0.05, Tukey s-b(k); n = 4).

Mentions: When using arbitrary genes to determine the relative expression levels of target genes in different samples the results can differ. For example, when using the most unstable gene for normalization, Hop transcript levels were higher in the nymphal stage compared to adult and pupae in the different development stages. However, after the best reference gene or the recommended two most stable references were used to normalize, no evident difference was detected (Fig. 4A). Similar results also occurred when calculating the relative expression levels of Hop after normalization with the unstable reference genes among the samples exposed to low temperatures (Fig. 4C). Therefore, it is important to determine the optimal reference genes for accurate normalization of qRT-PCR data, especially when differences in expression levels are subtle, as arbitrary selection of reference genes may decrease the accuracy of determining target gene expression. For example, the relative expression level of Hop showed no significant differences among the samples exposed to high temperatures when calculated using actin as the reference gene; however, the expression was significantly different when normalized by RPL32 and GAPDH (Fig. 4B). Therefore, in order to obtain accurate expression data, the expression stability of putative reference genes needs to be verified before each qRT-PCR experiment.


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)

Validation of reference gene selection.A) Relative expression levels of Hop in different developmental stages; B) Exposed to high temperatures; C) Exposed to low temperatures (P<0.05, Tukey s-b(k); n = 4).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111369-g004: Validation of reference gene selection.A) Relative expression levels of Hop in different developmental stages; B) Exposed to high temperatures; C) Exposed to low temperatures (P<0.05, Tukey s-b(k); n = 4).
Mentions: When using arbitrary genes to determine the relative expression levels of target genes in different samples the results can differ. For example, when using the most unstable gene for normalization, Hop transcript levels were higher in the nymphal stage compared to adult and pupae in the different development stages. However, after the best reference gene or the recommended two most stable references were used to normalize, no evident difference was detected (Fig. 4A). Similar results also occurred when calculating the relative expression levels of Hop after normalization with the unstable reference genes among the samples exposed to low temperatures (Fig. 4C). Therefore, it is important to determine the optimal reference genes for accurate normalization of qRT-PCR data, especially when differences in expression levels are subtle, as arbitrary selection of reference genes may decrease the accuracy of determining target gene expression. For example, the relative expression level of Hop showed no significant differences among the samples exposed to high temperatures when calculated using actin as the reference gene; however, the expression was significantly different when normalized by RPL32 and GAPDH (Fig. 4B). Therefore, in order to obtain accurate expression data, the expression stability of putative reference genes needs to be verified before each qRT-PCR experiment.

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