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

rationalMore 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.

objectivesThe 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.

methodsWe 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).

resultsgeNorm 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.

conclusionsOf 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.