Identification and Validation of Reference Genes for Gene Expression Analysis Using Quantitative PCR in Spodoptera Litura (Lepidoptera: Noctuidae)

Overview

Journal PLoS One

Specialties General Medicine
Science

Date 2013 Jul 23

PMID 23874494

Citations 80

Authors

Yanhui Lu

Miao Yuan

Xiwu Gao

Tinghao Kang

Sha Zhan

Hu Wan

Jianhong Li

Affiliations

Soon will be listed here.

Abstract

Reverse transcription quantitative polymerase chain reaction (qRT-PCR) has rapidly become the most sensitive and accurate method for the quantification of gene expression. To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable housekeeping genes is required. These housekeeping genes need to show stable expression under the given experimental conditions for the qRT-PCR results to be accurate. Unfortunately, there are no studies on the stability of housekeeping genes used in Spodoptera litura. In this study, eight candidate reference genes, elongation factor 1 alpha (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L10 (RPL10), ribosomal protein S3 (RPS3), beta actin (ACTB), beta FTZ-F1 (FTZF1), ubiquinol-cytochrome c reductase (UCCR), and arginine kinase (AK), were evaluated for their suitability as normalization genes under different experimental conditions using the statistical software programs, BestKeeper, geNorm and Normfinder, and the comparative ΔCt method. We determined the expression levels of the candidate reference genes for three biotic factors (developmental stage, tissue and population), and four abiotic treatments (temperature, insecticide, food and starvation). The results indicated that the best sets of candidates as reference genes were as follows: GAPDH and UCCR for developmental stages; RPL10, AK and EF1 for different tissues; RPL10 and EF1 for different populations in China; GAPDH and EF1 for temperature-stressed larvae; AK and ACTB for larvae treated with different insecticides; RPL10, GAPDH and UCCR for larvae fed different diets; RPS3 and ACTB for starved larvae. We believe that these results make an important contribution to gene analysis studies in S. litura and form the basis of further research on stable reference genes in S. litura and other organisms.

Citing Articles

The Selection and Validation of Reference Genes for RT-qPCR Analysis of the Predatory Natural Enemy (Hemiptera: Anthocoridae).

Wang C, Yin Z, Wang Y, Liu Y, Zhao S, Dai X Insects. 2025; 15(12.

PMID: 39769538 PMC: 11678729. DOI: 10.3390/insects15120936.

Reference genes to study the sex-biased expression of genes regulating Drosophila metabolism.

De Groef S, Ribeiro Lopes M, Winant M, Rosschaert E, Wilms T, Bolckmans L Sci Rep. 2024; 14(1):9518.

PMID: 38664539 PMC: 11045863. DOI: 10.1038/s41598-024-58863-5.

Evaluation of reference genes for quantitative expression analysis in (Coleoptera, Meloidae).

Shen C, Tang M, Li X, Zhu L, Li W, Deng P Front Physiol. 2024; 15:1345836.

PMID: 38651047 PMC: 11033477. DOI: 10.3389/fphys.2024.1345836.

Comprehensive Assessment of Reference Gene Expression within the Whitefly Using RT-qPCR.

Kong W, Lv X, Ran X, Mukangango M, Eric Derrick B, Qiu B Genes (Basel). 2024; 15(3).

PMID: 38540377 PMC: 10970672. DOI: 10.3390/genes15030318.

Validation and Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis in (Lepidoptera: Noctuidae).

Wang L, Yang C, Liu Q, Zhang X, Mei X, Zhang T Insects. 2024; 15(3).

PMID: 38535380 PMC: 10970824. DOI: 10.3390/insects15030185.

References

Paim R, Pereira M, Di Ponzio R, Rodrigues J, Guarneri A, Gontijo N . Validation of reference genes for expression analysis in the salivary gland and the intestine of Rhodnius prolixus (Hemiptera, Reduviidae) under different experimental conditions by quantitative real-time PCR. BMC Res Notes. 2012; 5:128. PMC: 3337225. DOI: 10.1186/1756-0500-5-128. View

Chen H, Yin Y, Li Y, Mahmud M, Wang Z . Identification and analysis of genes differentially expressed in the Spodoptera litura fat body in response to the biocontrol fungus, Nomuraea rileyi. Comp Biochem Physiol B Biochem Mol Biol. 2012; 163(2):203-10. DOI: 10.1016/j.cbpb.2012.05.018. 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

Chapuis M, Tohidi-Esfahani D, Dodgson T, Blondin L, Ponton F, Cullen D . Assessment and validation of a suite of reverse transcription-quantitative PCR reference genes for analyses of density-dependent behavioural plasticity in the Australian plague locust. BMC Mol Biol. 2011; 12:7. PMC: 3048552. DOI: 10.1186/1471-2199-12-7. View

Mamidala P, Rajarapu S, Jones S, Mittapalli O . Identification and validation of reference genes for quantitative real-time polymerase chain reaction in Cimex lectularius. J Med Entomol. 2011; 48(4):947-51. DOI: 10.1603/me10262. View

Walker N . Tech.Sight. A technique whose time has come. Science. 2002; 296(5567):557-9. DOI: 10.1126/science.296.5567.557. View

Thellin O, Zorzi W, Lakaye B, de Borman B, Coumans B, Hennen G . Housekeeping genes as internal standards: use and limits. J Biotechnol. 2000; 75(2-3):291-5. DOI: 10.1016/s0168-1656(99)00163-7. View

Radonic A, Thulke S, Mackay I, Landt O, Siegert W, Nitsche A . Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun. 2004; 313(4):856-62. DOI: 10.1016/j.bbrc.2003.11.177. View

Liang P, Gao X, Zheng B . Genetic basis of resistance and studies on cross-resistance in a population of diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae). Pest Manag Sci. 2003; 59(11):1232-6. DOI: 10.1002/ps.760. View

10.

Toutges M, Hartzer K, Lord J, Oppert B . Evaluation of reference genes for quantitative polymerase chain reaction across life cycle stages and tissue types of Tribolium castaneum. J Agric Food Chem. 2010; 58(16):8948-51. DOI: 10.1021/jf101603j. View

11.

Giulietti A, Overbergh L, Valckx D, Decallonne B, Bouillon R, Mathieu C . An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods. 2002; 25(4):386-401. DOI: 10.1006/meth.2001.1261. View

12.

Shen G, Jiang H, Wang X, Wang J . Evaluation of endogenous references for gene expression profiling in different tissues of the oriental fruit fly Bactrocera dorsalis (Diptera: Tephritidae). BMC Mol Biol. 2010; 11:76. PMC: 2972281. DOI: 10.1186/1471-2199-11-76. View

13.

Sun W, Jin Y, He L, Lu W, Li M . Suitable reference gene selection for different strains and developmental stages of the carmine spider mite, Tetranychus cinnabarinus, using quantitative real-time PCR. J Insect Sci. 2011; 10:208. PMC: 3029232. DOI: 10.1673/031.010.20801. View

14.

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

15.

Lu Y, Pang Y, Park Y, Gao X, Yao J, Zhang X . Genome organization, phylogenies, expression patterns, and three-dimensional protein models of two acetylcholinesterase genes from the red flour beetle. PLoS One. 2012; 7(2):e32288. PMC: 3281121. DOI: 10.1371/journal.pone.0032288. View

16.

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

17.

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

18.

Lee P, Sladek R, Greenwood C, Hudson T . Control genes and variability: absence of ubiquitous reference transcripts in diverse mammalian expression studies. Genome Res. 2002; 12(2):292-7. PMC: 155273. DOI: 10.1101/gr.217802. View

19.

Bear A, Simons A, Westerman E, Monteiro A . The genetic, morphological, and physiological characterization of a dark larval cuticle mutation in the butterfly, Bicyclus anynana. PLoS One. 2010; 5(7):e11563. PMC: 2904379. DOI: 10.1371/journal.pone.0011563. View

20.

Van Hiel M, Wielendaele P, Temmerman L, Van Soest S, Vuerinckx K, Huybrechts R . Identification and validation of housekeeping genes in brains of the desert locust Schistocerca gregaria under different developmental conditions. BMC Mol Biol. 2009; 10:56. PMC: 2700112. DOI: 10.1186/1471-2199-10-56. View