Identification and Evaluation of Suitable Reference Genes for Normalization of MicroRNA Expression in Helicoverpa Armigera (Lepidoptera: Noctuidae) Using Quantitative Real-Time PCR

Overview

Journal J Insect Sci

Specialty Biology

Date 2017 Mar 30

PMID 28355475

Citations 7

Authors

Yuhui Yang

Zhen Li

Jinjun Cao

Yanrong Li

Hui Li

Qingpo Yang

Qingwen Zhang

XiaoXia Liu

Affiliations

Soon will be listed here.

Abstract

More and more studies have focused on microRNAs (miRNAs) expression in the pest Helicoverpa armigera (Lepidoptera: Noctuidae) recently. Quantitative real-time PCR (qRT-PCR) is being widely used in miRNA expression studies. Suitable reference genes are necessary for the correct analysis of results. In this study, 10 candidate genes of H. armigera were selected and analyzed for their expression stability under different biotic and abiotic conditions with 3 statistical methods, including geNorm, NormFinder, and Bestkeeper. Combination the best number of reference genes was calculated by geNorm. One target gene, let-7, was used to validate the selection of reference genes. The suitable candidate reference genes were shown as follows: miR-9 and U6 snRNA for developmental stages, miR-100 and U6 snRNA for larval tissues, miR-100 and miR-305 for adult tissues, miR-9 and miR-279 for parasitic treatment, miR-998 and U6 snRNA for nuclear polyhedrosis virus infection, miR-9 and U6 snRNA for insecticide treatment, miR-92a, miR-100, and miR-279 for temperature treatment, miR-92a, miR-305, and miR-998 for starvation treatment, miR-9 and miR-279 for light treatment, miR-305 and miR-998 for hormone treatment, and there was not one reference gene suitable for all samples. This study could promote future research on miRNAs expression in H. armigera with optimal reference genes under different experimental conditions.

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References

Zhang J, Zhang S, Han S, Wu T, Li X, Li W . Genome-wide identification of microRNAs in larch and stage-specific modulation of 11 conserved microRNAs and their targets during somatic embryogenesis. Planta. 2012; 236(2):647-57. DOI: 10.1007/s00425-012-1643-9. View

Dhaene B, Mestdagh P, Hellemans J, Vandesompele J . miRNA expression profiling: from reference genes to global mean normalization. Methods Mol Biol. 2011; 822:261-72. DOI: 10.1007/978-1-61779-427-8_18. View

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

Lin Y, Lai Z . Evaluation of suitable reference genes for normalization of microRNA expression by real-time reverse transcription PCR analysis during longan somatic embryogenesis. Plant Physiol Biochem. 2013; 66:20-5. DOI: 10.1016/j.plaphy.2013.02.002. View

Asgari S . MicroRNA functions in insects. Insect Biochem Mol Biol. 2012; 43(4):388-97. DOI: 10.1016/j.ibmb.2012.10.005. View

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

Kim V, Han J, Siomi M . Biogenesis of small RNAs in animals. Nat Rev Mol Cell Biol. 2009; 10(2):126-39. DOI: 10.1038/nrm2632. View

Zhang S, Li Z, Nian X, Wu F, Shen Z, Zhang B . Sequence analysis, expression profiles and function of thioredoxin 2 and thioredoxin reductase 1 in resistance to nucleopolyhedrovirus in Helicoverpa armigera. Sci Rep. 2015; 5:15531. PMC: 4621414. DOI: 10.1038/srep15531. View

Liang P, Feng B, Zhou X, Gao X . Identification and developmental profiling of microRNAs in diamondback moth, Plutellaxylostella (L.). PLoS One. 2013; 8(11):e78787. PMC: 3827265. DOI: 10.1371/journal.pone.0078787. View

10.

Feng H, Huang X, Zhang Q, Wei G, Wang X, Kang Z . Selection of suitable inner reference genes for relative quantification expression of microRNA in wheat. Plant Physiol Biochem. 2011; 51:116-22. DOI: 10.1016/j.plaphy.2011.10.010. View

11.

Wu K, Lu Y, Feng H, Jiang Y, Zhao J . Suppression of cotton bollworm in multiple crops in China in areas with Bt toxin-containing cotton. Science. 2008; 321(5896):1676-8. DOI: 10.1126/science.1160550. View

12.

Agrawal N, Sachdev B, Rodrigues J, Sree K, Bhatnagar R . Development associated profiling of chitinase and microRNA of Helicoverpa armigera identified chitinase repressive microRNA. Sci Rep. 2013; 3:2292. PMC: 3724176. DOI: 10.1038/srep02292. View

13.

Haller F, Kulle B, Schwager S, Gunawan B, von Heydebreck A, Sultmann H . Equivalence test in quantitative reverse transcription polymerase chain reaction: confirmation of reference genes suitable for normalization. Anal Biochem. 2004; 335(1):1-9. DOI: 10.1016/j.ab.2004.08.024. View

14.

Bustin S, Beaulieu J, Huggett J, Jaggi R, Kibenge F, Olsvik P . MIQE précis: Practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments. BMC Mol Biol. 2010; 11:74. PMC: 2955025. DOI: 10.1186/1471-2199-11-74. View

15.

Mallona I, Lischewski S, Weiss J, Hause B, Egea-Cortines M . Validation of reference genes for quantitative real-time PCR during leaf and flower development in Petunia hybrida. BMC Plant Biol. 2010; 10:4. PMC: 2827423. DOI: 10.1186/1471-2229-10-4. View

16.

Mahoney D, Carey K, Fu M, Snow R, Cameron-Smith D, Parise G . Real-time RT-PCR analysis of housekeeping genes in human skeletal muscle following acute exercise. Physiol Genomics. 2004; 18(2):226-31. DOI: 10.1152/physiolgenomics.00067.2004. View

17.

Wang X, Kang L . Rapid cold hardening in young hoppers of the migratory locust Locusta migratoria L. (Orthoptera: Acridiidae). Cryo Letters. 2003; 24(5):331-40. View

18.

Lee R, Feinbaum R, Ambros V . The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell. 1993; 75(5):843-54. DOI: 10.1016/0092-8674(93)90529-y. View

19.

Kang K, Peng X, Luo J, Gou D . Identification of circulating miRNA biomarkers based on global quantitative real-time PCR profiling. J Anim Sci Biotechnol. 2012; 3(1):4. PMC: 3415128. DOI: 10.1186/2049-1891-3-4. View

20.

Ling L, Ge X, Li Z, Zeng B, Xu J, Aslam A . MicroRNA Let-7 regulates molting and metamorphosis in the silkworm, Bombyx mori. Insect Biochem Mol Biol. 2014; 53:13-21. DOI: 10.1016/j.ibmb.2014.06.011. View