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Exploring valid reference genes for quantitative real-time PCR analysis in Plutella xylostella (Lepidoptera: Plutellidae).

ABSTRACT: Quantitative real-time PCR (qRT-PCR), a primary tool in gene expression analysis, requires an appropriate normalization strategy to control for variation among samples. The best option is to compare the mRNA level of a target gene with that of reference gene(s) whose expression level is stable across various experimental conditions. In this study, expression profiles of eight candidate reference genes from the diamondback moth, Plutella xylostella, were evaluated under diverse experimental conditions. RefFinder, a web-based analysis tool, integrates four major computational programs including geNorm, Normfinder, BestKeeper, and the comparative ?Ct method to comprehensively rank the tested candidate genes. Elongation factor 1 (EF1) was the most suited reference gene for the biotic factors (development stage, tissue, and strain). In contrast, although appropriate reference gene(s) do exist for several abiotic factors (temperature, photoperiod, insecticide, and mechanical injury), we were not able to identify a single universal reference gene. Nevertheless, a suite of candidate reference genes were specifically recommended for selected experimental conditions. Our finding is the first step toward establishing a standardized qRT-PCR analysis of this agriculturally important insect pest.

SUBMITTER: Fu W

PROVIDER: S-EPMC3753443 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Exploring valid reference genes for quantitative real-time PCR analysis in Plutella xylostella (Lepidoptera: Plutellidae).

Fu Wei W Xie Wen W Zhang Zhuo Z Wang Shaoli S Wu Qingjun Q Liu Yong Y Zhou Xiaomao X Zhou Xuguo X Zhang Youjun Y

International journal of biological sciences 20130820 8

Quantitative real-time PCR (qRT-PCR), a primary tool in gene expression analysis, requires an appropriate normalization strategy to control for variation among samples. The best option is to compare the mRNA level of a target gene with that of reference gene(s) whose expression level is stable across various experimental conditions. In this study, expression profiles of eight candidate reference genes from the diamondback moth, Plutella xylostella, were evaluated under diverse experimental condi ...[more]

PMID: 23983612

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Project description:BackgroundNorcantharidin (NCTD), a demethylated derivative of cantharidin (defensive toxin of blister beetles), has been reported to exhibit insecticidal activity against various types of agricultural pests. However, NCTD applications are limited by its poor water solubility and high dosage requirement. Nanoemulsions have attracted much attentions due to the transparent or translucence appearance, physical stability, high bioavailability and non-irritant in nature. In general, nanoemulsions with small droplet size can enhance the bioavailability of drugs, whereas this phenomenon is likely system dependent. In present study, NCTD nanoemulsions were developed and optimized to evaluate and improve the insecticidal activity of NCTD against Plutella xylostella (Lepidotera: Plutellidae) by a spontaneous emulsification method.ResultsTriacetin, Cremophor EL and butanol were selected as the constituents of NCTD nanoemulsions via solubility determination, emulsification efficiency and ternary phase diagram construction. Insecticidal activity of NCTD nanoemulsion was associated with the content of surfactant and cosurfactant: (1) Higher effective toxicity exhibited at Smix (surfactant to cosurfactant mass ratio) = 3:1 that may be associated with the changes in interfacial tension; (2) NCTD nanoemulsion at 3:7 < SOR (surfactant to oil mass ratio) < 6:4 was more effective at lower surfactant level, which was attributed to the relatively slow diffusion rate of NCTD hindering by excess surfactant. Interestingly, nanoemulsions with smaller droplets were not found to be more effective in our study.ConclusionsThe optimized NCTD nanoemulsion (triacetin/Cremophor EL/butanol (60/20/20, w/w)) exhibited effective insecticidal activity (LC50 60.414 mg/l, LC90 185.530 mg/l, 48 h) than the NCTD acetone solution (LC50 175.602 mg/L, LC90 303.050 mg/L, 48 h). Spontaneous emulsifying nanoemulsion employed to formulate this poor water-soluble pesticide is a potential system for agriculture application.

Dataset Information

Exploring valid reference genes for quantitative real-time PCR analysis in Plutella xylostella (Lepidoptera: Plutellidae).

Publications

Exploring valid reference genes for quantitative real-time PCR analysis in Plutella xylostella (Lepidoptera: Plutellidae).

Similar Datasets

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