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

Abstract

Rational 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, qRT-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. Objectives The primary aim was to evaluate the stability of 16 reference gene candidates in C. elegans exposed to nicotine and their two subsequent generations for determining the most reliable reference genes for multigenerational study. Methods 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, ΔCt method, NormFinder, BestKeeper, and ReFinder). Results 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 MDH-2 may serve the top reliable reference genes. Comparative ΔCt 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. Conclusions 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.

Drug-dependent behaviors and nicotinic acetylcholine receptor expressions in Caenorhabditis elegans following chronic nicotine exposure

“…four independent experiments. In qPCR, F35G12.2, a conserved house-keeping gene that encodes a C. elegans ortholog of mitochondrial NAD+:isocitrate dehydrogenase, was employed as the reference gene for normalizing qPCR results (Taki & Zhang, 2013). The results were analyzed using the ΔΔCt method (Livak & Schmittgen, 2001).…”

The Selection of Reliable Reference Genes for RT-qPCR Analysis of Anisakis simplex Sensu Stricto Gene Expression from Different Developmental Stages

“…Trivedi and Arasu [ 26 ], who identified reference genes for A. caninum, and Li et al [ 27 ], who analyzed B. malayi, reported that actin was the best endogenous control for the investigated organisms. Taki and Zhang [ 28 ] validated the stability of the reference genes for C. elegans and concluded that ef-1α was characterized by sufficiently stable expression to be used as a reference gene. In an analysis of gene expression in C. elegans , Hoogewijs et al [ 29 ] identified actin and gapdh as genes with the least stable expression, which is consistent with the results noted for A. simplex (Fig.…”

Transcriptome-wide selection of a reliable set of reference genes for gene expression studies in potato cyst nematodes (Globodera spp.)

“…A set of 15 genes previously used as references in the model nematode C . elegans [ 3 , 19 , 20 ] were selected to be evaluated in G . rostochiensis .…”