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, 9 (1), e86503
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Selection and Evaluation of Potential Reference Genes for Gene Expression Analysis in the Brown Planthopper, Nilaparvata Lugens (Hemiptera: Delphacidae) Using Reverse-Transcription Quantitative PCR

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Selection and Evaluation of Potential Reference Genes for Gene Expression Analysis in the Brown Planthopper, Nilaparvata Lugens (Hemiptera: Delphacidae) Using Reverse-Transcription Quantitative PCR

Miao Yuan et al. PLoS One.

Abstract

The brown planthopper (BPH), Nilaparvata lugens (Hemiptera, Delphacidae), is one of the most important rice pests. Abundant genetic studies on BPH have been conducted using reverse-transcription quantitative real-time PCR (qRT-PCR). Using qRT-PCR, the expression levels of target genes are calculated on the basis of endogenous controls. These genes need to be appropriately selected by experimentally assessing whether they are stably expressed under different conditions. However, such studies on potential reference genes in N. lugens are lacking. In this paper, we presented a systematic exploration of eight candidate reference genes in N. lugens, namely, actin 1 (ACT), muscle actin (MACT), ribosomal protein S11 (RPS11), ribosomal protein S15e (RPS15), alpha 2-tubulin (TUB), elongation factor 1 delta (EF), 18S ribosomal RNA (18S), and arginine kinase (AK) and used four alternative methods (BestKeeper, geNorm, NormFinder, and the delta Ct method) to evaluate the suitability of these genes as endogenous controls. We examined their expression levels among different experimental factors (developmental stage, body part, geographic population, temperature variation, pesticide exposure, diet change, and starvation) following the MIQE (Minimum Information for publication of Quantitative real time PCR Experiments) guidelines. Based on the results of RefFinder, which integrates four currently available major software programs to compare and rank the tested candidate reference genes, RPS15, RPS11, and TUB were found to be the most suitable reference genes in different developmental stages, body parts, and geographic populations, respectively. RPS15 was the most suitable gene under different temperature and diet conditions, while RPS11 was the most suitable gene under different pesticide exposure and starvation conditions. This work sheds light on establishing a standardized qRT-PCR procedure in N. lugens, and serves as a starting point for screening for reference genes for expression studies of related insects.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Expression levels of candidate reference genes.
The expression level of candidate N. lugens reference genes in the total samples is shown in terms of the cycle threshold number (Ct-value). The data are expressed as whisker box plots; the box represents the 25th–75th percentiles, the median is indicated by a bar across the box, the whiskers on each box represent the minimum and maximum values.
Figure 2
Figure 2. Determination of the optimal number of reference genes for accurate normalization calculated by geNorm.
The value of Vn/Vn+1 indicates the pairwise variation (Y axis) between two sequential normalization factors and determines the optimal number of reference genes required for accurate normalization. A value below 0.15 indicates that an additional reference gene will not significantly improve normalization.

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Grant support

This research was supported by China Hubei Province Science & Technology Department (No. 2009BFA011). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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