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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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Expression levels of candidate reference genes in different samples.The black dot indicates the mean of duplicate samples, and the bars indicate the standard deviation of the mean.
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pone-0111369-g001: Expression levels of candidate reference genes in different samples.The black dot indicates the mean of duplicate samples, and the bars indicate the standard deviation of the mean.

Mentions: Expression levels were determined as the number of cycles needed for amplification to reach a fixed threshold in the exponential phase of the qPCR reaction [21]. Except for 18S, the gene expression analysis of all seven candidate reference genes displayed a narrow range of mean Ct values across all experimental samples (Fig. 1). The mean Ct values of the seven reference genes varied from 9.62 (18S) to 26.54 (actin). The 18S gene showed the most abundant expression levels, followed by RPL32 (mean Ct 20.49), EF-1(mean Ct 21.67), tubulin (mean Ct 22.67), GAPDH (mean Ct 23.12), and H3 (mean Ct 24.35). The smallest Ct variation among the experimental samples was EF-1 with a value of 4.01, while actin had the highest Ct variation with a value of 7.74. All candidate genes exhibited relatively small variation in Ct values.


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)

Expression levels of candidate reference genes in different samples.The black dot indicates the mean of duplicate samples, and the bars indicate the standard deviation of the mean.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111369-g001: Expression levels of candidate reference genes in different samples.The black dot indicates the mean of duplicate samples, and the bars indicate the standard deviation of the mean.
Mentions: Expression levels were determined as the number of cycles needed for amplification to reach a fixed threshold in the exponential phase of the qPCR reaction [21]. Except for 18S, the gene expression analysis of all seven candidate reference genes displayed a narrow range of mean Ct values across all experimental samples (Fig. 1). The mean Ct values of the seven reference genes varied from 9.62 (18S) to 26.54 (actin). The 18S gene showed the most abundant expression levels, followed by RPL32 (mean Ct 20.49), EF-1(mean Ct 21.67), tubulin (mean Ct 22.67), GAPDH (mean Ct 23.12), and H3 (mean Ct 24.35). The smallest Ct variation among the experimental samples was EF-1 with a value of 4.01, while actin had the highest Ct variation with a value of 7.74. All candidate genes exhibited relatively small variation in Ct values.

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