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Determination of reliable reference genes for multi-generational gene expression analysis on C. elegans exposed to abused drug nicotine.

ABSTRACT: More research has recently been focused on multigenerational toxicogenomics impacts. Such studies rely on behavioral as well as genetic and epigenetic analyses using various biotechniques. Of these technologies, quantitative reverse transcriptase PCR is considered as a mature discovery and validation tool. Nevertheless, the interpretation of the resulting gene expression necessitates the establishment of reliable internal controls for normalization. No study has been performed to identify reliable reference genes in multigenerational settings.The primary aim was to evaluate the stability of 16 reference gene candidates in Caenorhabditis elegans exposed to nicotine and their two subsequent generations for determining the most reliable reference genes for multigenerational study.We exposed C. elegans to nicotine in the F0 generation and investigated the relative stabilities of 16 housekeeping genes in L4 larvae across three generations (F0, F1, and F2) using five statistical approaches (geNorm, ?Ct method, NormFinder, BestKeeper, and RefFinder).geNorm shows that CDC-42 and Y45F10D.4 were the most stable reference genes. Based on NormFinder, TBA-1, EIF3.C, ARP-6, CDC-42, and MDH2 may serve as the top reliable reference genes. Comparative ?Ct method ranked TBA-1, CDC-42, EIF3.C, ARP-6, and Y45F10D.4 as the most stable reference genes. BestKeeper shows that Y45F10D.4, F35G12.2, TBA-1, CDC-42, and CSQ-1were better reference genes. Overall, TBA-1, CDC-42, EIF3.C, ARP-6, and Y45F10D.4 were the most reliable reference genes for mutigenerational nicotine-exposed study.Of the 16 tested gene candidates, TBA-1 and CDC-42 were the two most stable reference genes for performing reliable gene expression normalization in the multigenerational impact of nicotine exposure.

SUBMITTER: Taki FA

PROVIDER: S-EPMC3795882 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Determination of reliable reference genes for multi-generational gene expression analysis on C. elegans exposed to abused drug nicotine.

Taki Faten A FA Zhang Baohong B

Psychopharmacology 20130517 1

<h4>Rational</h4>More research has recently been focused on multigenerational toxicogenomics impacts. Such studies rely on behavioral as well as genetic and epigenetic analyses using various biotechniques. Of these technologies, quantitative reverse transcriptase PCR is considered as a mature discovery and validation tool. Nevertheless, the interpretation of the resulting gene expression necessitates the establishment of reliable internal controls for normalization. No study has been performed t ...[more]

PMID: 23681163

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Project description:Nicotine, the major psychoactive compound in tobacco, targets nicotinic acetylcholine receptors (nAChRs) and results in drug dependence. The nematode Caenorhabditis elegans' (C. elegans) genome encodes conserved and extensive nicotinic receptor subunits, representing a useful system to investigate nicotine-induced nAChR expressions in the context of drug dependence. However, the in vivo expression pattern of nAChR genes under chronic nicotine exposure has not been fully investigated. To define the role of nAChR genes involved in nicotine-induced locomotion changes and the development of tolerance to these effects, we characterized the locomotion behavior combining the use of two systems: the Worm Tracker hardware and the WormLab software. Our results indicate that the combined system is an advantageous alternative to define drug-dependent locomotion behavior in C. elegans. Chronic (24-h dosing) nicotine exposure at 6.17 and 61.7μM induced nicotine-dependent behaviors, including drug stimulation, tolerance/adaption, and withdrawal responses. Specifically, the movement speed of naïve worms on nicotine-containing environments was significantly higher than on nicotine-free environments, suggesting locomotion stimulation by nicotine. In contrast, the 24-h 6.17μM nicotine-treated worms exhibited significantly higher speeds on nicotine-free plates than on nicotine-containing plates. Furthermore significantly increased locomotion behavior during nicotine cessation was observed in worms treated with a higher nicotine concentration of 61.7μM. The relatively low locomotion speed of nicotine-treated worms on nicotine-containing environments also indicates adaption/tolerance of worms to nicotine following chronic nicotine exposure. In addition, this study provides useful information regarding the comprehensive in vivo expression profile of the 28 "core" nAChRs following different dosages of chronic nicotine treatments. Eleven genes (lev-1, acr-6, acr-7, acr-11, lev-8, acr-14, acr-16, acr-20, acr-21, ric-3, and unc-29) were significantly up-regulated following 61.7μM nicotine treatment, in which worms showed significantly increased locomotion behavior. This study provides insights into the linkage between nicotine-induced locomotion behavior and the regulation of nicotinic acetylcholine receptors.

Dataset Information

Determination of reliable reference genes for multi-generational gene expression analysis on C. elegans exposed to abused drug nicotine.

Publications

Determination of reliable reference genes for multi-generational gene expression analysis on C. elegans exposed to abused drug nicotine.

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