Selection of Reference Genes for RT-qPCR Analysis in the Monarch Butterfly, Danaus Plexippus (L.), a Migrating Bio-Indicator

Overview

Journal PLoS One

Specialties General Medicine
Science

Date 2015 Jun 2

PMID 26030778

Citations 34

Authors

Huipeng Pan

Xiaowei Yang

Keith Bidne

Richard L Hellmich

Blair D Siegfried

Xuguo Zhou

Affiliations

Soon will be listed here.

Abstract

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a powerful technique to quantify gene expression. To facilitate gene expression study and obtain accurate results, normalization relative to stably expressed reference genes is crucial. The monarch butterfly, Danaus plexippus (L.), is one of the most recognized insect species for its spectacular annual migration across North America. Besides its great voyages, D. plexippus has drawn attention to its role as a bio-indicator, ranging from genetically modified organisms (GMOs) to natural ecosystems. In this study, nine reference genes from D. plexippus genome were selected as the candidate reference genes. The expression profiles of these candidates under various biotic and abiotic conditions were evaluated using the four readily available computational programs, BestKeeper, Normfinder, geNorm, and ΔCt method, respectively. Moreover, RefFinder, a web-based computational platform integrating the four above mentioned algorisms, provided a comprehensive ranking of the stability of these reference genes. As a result, a suite of reference genes were recommended for each experimental condition. Specifically, elongation factor 1α (EF1A) and ribosomal protein 49 (RP49) were the most stable reference genes, respectively, under biotic (development, tissue, and sex) and abiotic (photoperiod, temperature, and dietary RNAi) conditions. With the recent release of a 273-million base pair draft genome, results from this study allow us to establish a standardized RT-qPCR analysis and lay a foundation for the subsequent genomic and functional genomic research in D. plexippus, a major bio-indicator and an emerging model for migratory animals.

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References

Huvenne H, Smagghe G . Mechanisms of dsRNA uptake in insects and potential of RNAi for pest control: a review. J Insect Physiol. 2009; 56(3):227-35. DOI: 10.1016/j.jinsphys.2009.10.004. View

Jacob F, Guertler R, Naim S, Nixdorf S, Fedier A, Hacker N . Careful selection of reference genes is required for reliable performance of RT-qPCR in human normal and cancer cell lines. PLoS One. 2013; 8(3):e59180. PMC: 3598660. DOI: 10.1371/journal.pone.0059180. View

Hornakova D, Matouskova P, Kindl J, Valterova I, Pichova I . Selection of reference genes for real-time polymerase chain reaction analysis in tissues from Bombus terrestris and Bombus lucorum of different ages. Anal Biochem. 2009; 397(1):118-20. DOI: 10.1016/j.ab.2009.09.019. View

Yang C, Pan H, Liu Y, Zhou X . Selection of reference genes for expression analysis using quantitative real-time PCR in the pea aphid, Acyrthosiphon pisum (Harris) (Hemiptera, Aphidiae). PLoS One. 2014; 9(11):e110454. PMC: 4244036. DOI: 10.1371/journal.pone.0110454. View

Li R, Xie W, Wang S, Wu Q, Yang N, Yang X . Reference gene selection for qRT-PCR analysis in the sweetpotato whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae). PLoS One. 2013; 8(1):e53006. PMC: 3540095. DOI: 10.1371/journal.pone.0053006. View

Burand J, Hunter W . RNAi: future in insect management. J Invertebr Pathol. 2012; 112 Suppl:S68-74. DOI: 10.1016/j.jip.2012.07.012. View

Hudson M . Sequencing breakthroughs for genomic ecology and evolutionary biology. Mol Ecol Resour. 2011; 8(1):3-17. DOI: 10.1111/j.1471-8286.2007.02019.x. View

Woods J, Wilson J, Runkle J . Influence of climate on butterfly community and population dynamics in Western Ohio. Environ Entomol. 2008; 37(3):696-706. DOI: 10.1603/0046-225x(2008)37[696:iocobc]2.0.co;2. View

Pfaffl M, Tichopad A, Prgomet C, Neuvians T . Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper--Excel-based tool using pair-wise correlations. Biotechnol Lett. 2004; 26(6):509-15. DOI: 10.1023/b:bile.0000019559.84305.47. View

10.

Bustin S, Benes V, Nolan T, Pfaffl M . Quantitative real-time RT-PCR--a perspective. J Mol Endocrinol. 2005; 34(3):597-601. DOI: 10.1677/jme.1.01755. View

11.

Mao Y, Cai W, Wang J, Hong G, Tao X, Wang L . Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol. Nat Biotechnol. 2007; 25(11):1307-13. DOI: 10.1038/nbt1352. View

12.

Livak K, Schmittgen T . Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2002; 25(4):402-8. DOI: 10.1006/meth.2001.1262. View

13.

Guo J, Ling H, Wu Q, Xu L, Que Y . The choice of reference genes for assessing gene expression in sugarcane under salinity and drought stresses. Sci Rep. 2014; 4:7042. PMC: 4229666. DOI: 10.1038/srep07042. View

14.

Sears M, Hellmich R, Oberhauser K, Pleasants J, Mattila H, Siegfried B . Impact of Bt corn pollen on monarch butterfly populations: a risk assessment. Proc Natl Acad Sci U S A. 2001; 98(21):11937-42. PMC: 59819. DOI: 10.1073/pnas.211329998. View

15.

Silver N, Best S, Jiang J, Thein S . Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR. BMC Mol Biol. 2006; 7:33. PMC: 1609175. DOI: 10.1186/1471-2199-7-33. View

16.

Andersen C, Jensen J, Orntoft T . Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res. 2004; 64(15):5245-50. DOI: 10.1158/0008-5472.CAN-04-0496. View

17.

Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A . Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002; 3(7):RESEARCH0034. PMC: 126239. DOI: 10.1186/gb-2002-3-7-research0034. View

18.

Ponton F, Chapuis M, Pernice M, Sword G, Simpson S . Evaluation of potential reference genes for reverse transcription-qPCR studies of physiological responses in Drosophila melanogaster. J Insect Physiol. 2011; 57(6):840-50. DOI: 10.1016/j.jinsphys.2011.03.014. View

19.

Stensmyr M, Hansson B . A genome befitting a monarch. Cell. 2011; 147(5):970-2. DOI: 10.1016/j.cell.2011.11.009. View

20.

Zhu X, Yuan M, Shakeel M, Zhang Y, Wang S, Wang X . Selection and evaluation of reference genes for expression analysis using qRT-PCR in the beet armyworm Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae). PLoS One. 2014; 9(1):e84730. PMC: 3893131. DOI: 10.1371/journal.pone.0084730. View