Dataset Information

Selection of reference genes for qRT-PCR and expression analysis of high-altitude-related genes in grassland caterpillars (Lepidoptera: Erebidae: Gynaephora) along an altitude gradient.

ABSTRACT: Changes in gene expression patterns can reflect the adaptation of organisms to divergent environments. Quantitative real-time PCR (qRT-PCR) is an important tool for ecological adaptation studies at the gene expression level. The quality of the results of qRT-PCR analysis largely depends on the availability of reliable reference genes (RGs). To date, reliable RGs have not been determined for adaptive evolution studies in insects using a standard approach. Here, we evaluated the reliability of 17 candidate RGs for five Gynaephora populations inhabiting various altitudes of the Tibetan Plateau (TP) using four independent (geNorm, NormFinder, BestKeeper, and the deltaCt method) and one comprehensive (RefFinder) algorithms. Our results showed that EF1-?, RPS15, and RPS13 were the top three most suitable RGs, and a combination of these three RGs was the most optimal for normalization. Conversely, RPS2,ACT, and RPL27 were the most unstable RGs. The expression profiles of two target genes (HSP70 and HSP90) were used to confirm the reliability of the chosen RGs. Additionally, the expression patterns of four other genes (GPI,HIF1A,HSP20, and USP) associated with adaptation to extreme environments were assessed to explore the adaptive mechanisms of TP Gynaephora species to divergent environments. Each of these six target genes showed discrepant expression patterns among the five populations, suggesting that the observed expression differences may be associated with the local adaptation of Gynaephora to divergent altitudinal environments. This study is a useful resource for studying the adaptive evolution of TP Gynaephora to divergent environments using qRT-PCR, and it also acts as a guide for selecting suitable RGs for ecological and evolutionary studies in insects.

SUBMITTER: Zhang L

PROVIDER: S-EPMC5677504 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Publications

Selection of reference genes for qRT-PCR and expression analysis of high-altitude-related genes in grassland caterpillars (Lepidoptera: Erebidae: <i>Gynaephora</i>) along an altitude gradient.

Zhang Li L Zhang Qi-Lin QL Wang Xiao-Tong XT Yang Xing-Zhuo XZ Li Xiao-Peng XP Yuan Ming-Long ML

Ecology and evolution 20170925 21

Changes in gene expression patterns can reflect the adaptation of organisms to divergent environments. Quantitative real-time PCR (qRT-PCR) is an important tool for ecological adaptation studies at the gene expression level. The quality of the results of qRT-PCR analysis largely depends on the availability of reliable reference genes (RGs). To date, reliable RGs have not been determined for adaptive evolution studies in insects using a standard approach. Here, we evaluated the reliability of 17 ...[more]

PMID: 29152197

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Project description:Quantitative real-time PCR (qRT-PCR) is a reliable and reproducible technique for measuring and evaluating changes in gene expression. The most common method for analyzing qRT-PCR data is to normalize mRNA levels of target genes to internal reference genes. Evaluating and selecting stable reference genes on a case-by-case basis is critical. The present study aimed to facilitate gene expression studies by identifying the most suitable reference genes for normalization of mRNA expression in qRT-PCR analysis of the beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae). For this purpose, three software tools (geNorm, NormFinder and BestKeeper) were used to investigate 10 candidate reference genes in nine developmental stages and five different tissues (epidermis, head, midgut, fat body and hemolymph) in three larval physiological stages (molting, feeding and wandering stages) of, S. exigua. With the exception of 18S ribosomal RNA (18S), all other candidate genes evaluated, β-actin1(ACT1), β-actin2 (ACT2), elongation factor1(EF1), elongation factor 2 (EF2), Glyceralde hyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L10 (L10), ribosomal protein L17A (L17A), superoxide dismutase (SOD), α-tubulin (TUB),proved to be acceptable reference genes. However, their suitability partly differed between physiological stages and different tissues. L10, EF2 and L17A ranked highest in all tissue sample sets. SOD, ACT2, GAPDH, EF1 and ACT1 were stably expressed in all developmental stage sample sets; ACT2, ACT1 and L10 for larvae sample sets; GAPDH, ACT1 and ACT2 for pupae and adults; SOD and L17A for males; and EF2 and SOD for females. The expression stability of genes varied in different conditions. The findings provided here demonstrated, with a few exceptions, the suitability of most of the 10 reference genes tested in tissues and life developmental stages. Overall, this study emphasizes the importance of validating reference genes for qRT-PCR analysis in S. exigua.

Dataset Information

Selection of reference genes for qRT-PCR and expression analysis of high-altitude-related genes in grassland caterpillars (Lepidoptera: Erebidae: Gynaephora) along an altitude gradient.

Publications

Selection of reference genes for qRT-PCR and expression analysis of high-altitude-related genes in grassland caterpillars (Lepidoptera: Erebidae: <i>Gynaephora</i>) along an altitude gradient.

Similar Datasets

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