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

Lu, Yanhui; Yuan, Miao; Gao, Xiwu; Kang, Tinghao; Zhan, Sha; Wan, Hu; Li, Jianhong
July 2013
PLoS ONE;Jul2013, Vol. 8 Issue 7, p1
Academic Journal
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.


Related Articles

  • Housekeeping gene expression stability in reproductive tissues after mitogen stimulation. Arenas-Hernandez, Marcia; Vega-Sanchez, Rodrigo // BMC Research Notes;2013, Vol. 6 Issue 1, p1 

    Background: Intrauterine infection during pregnancy can trigger a local inflammatory response leading to several complications, such as preterm labor. Many studies have used in vitro and in vivo models employing mitogens to induce the expression of the characteristic proinflammatory mediators...

  • Validation of reference genes in Penicillium echinulatum to enable gene expression study using real-time quantitative RT-PCR. Zampieri, Denise; Nora, Luísa; Basso, Vanessa; Camassola, Marli; Dillon, Aldo // Current Genetics;Aug2014, Vol. 60 Issue 3, p231 

    Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is a methodology that facilitates the quantification of mRNA expression in a given sample. Analysis of relative gene expression by qRT-PCR requires normalization of the data using a reference gene that is expressed at a...

  • Histological evidence: housekeeping genes beta-actin and GAPDH are of limited value for normalization of gene expression. Lin, Juntang; Redies, Christoph // Development Genes & Evolution;Nov2012, Vol. 222 Issue 6, p369 

    Housekeeping genes are widely used as internal controls for gene expression normalization for western blotting, northern blotting, RT-PCR, etc. They are generally thought to be expressed in all cells of the organism at similar levels because it is assumed that these genes are required for the...

  • Identification of appropriate reference genes for human mesenchymal stem cell analysis by quantitative real-time PCR. Li, Xiuying; Yang, Qiwei; Bai, Jinping; Xuan, Yali; Wang, Yimin // Biotechnology Letters;Jan2015, Vol. 37 Issue 1, p67 

    Normalization to a reference gene is the method of choice for quantitative reverse transcription-PCR (RT-qPCR) analysis. The stability of reference genes is critical for accurate experimental results and conclusions. We have evaluated the expression stability of eight commonly used reference...

  • GAPDH Pseudogenes and the Quantification of Feline Genomic DNA Equivalents. Helfer-Hungerbuehler, A. Katrin; Widmer, Stefan; Hofmann-Lehmann, Regina // Molecular Biology International;2013, p1 

    Quantitative real-time PCR (qPCR) is broadly used to detect and quantify nucleic acid targets. In order to determine cell copy number and genome equivalents, a suitable reference gene that is present in a defined number in the genome is needed, preferably as a single copy gene. Formost...

  • Validation of reference genes for gene expression studies in peanut by quantitative real-time RT-PCR. Chi, Xiaoyuan; Hu, Ruibo; Yang, Qingli; Zhang, Xiaowen; Pan, Lijuan; Chen, Na; Chen, Mingna; Yang, Zhen; Wang, Tong; He, Yanan; Yu, Shanlin // Molecular Genetics & Genomics;Feb2012, Vol. 287 Issue 2, p167 

    Quantitative real-time reverse transcription PCR (qRT-PCR), a sensitive technique for quantifying gene expression, depends on the stability of the reference gene(s) used for data normalization. Only a few studies on the reference genes have been done with peanut to date. In the present study, 14...

  • Cloning and characterization of two allelic glyceraldehyde-3-phosphate dehydrogenase genes in Auricularia auricula- judae. Fan, Xiuzhi; Zhou, Yan; Xiao, Yang; Bian, Yinbing // World Journal of Microbiology & Biotechnology;Jan2014, Vol. 30 Issue 1, p181 

    Two allelic variants of the gpd gene, Gpd and Gpd, were isolated based on a putative glyceraldehyde-3-phosphate dehydrogenase encoding sequence from the transcriptome of Auricularia auricula- judae strain Au916. The two alleles were found to have a 73 bp length discrepancy and 39 SNP variations....

  • Quantification of Hantaan Virus with a SYBR Green â… -Based One-Step qRT-PCR Assay. Jiang, Wei; Yu, Hai-tao; Zhao, Ke; Zhang, Ye; Du, Hong; Wang, Ping-zhong; Bai, Xue-fan // PLoS ONE;Nov2013, Vol. 8 Issue 11, p1 

    Hantaan virus (HTNV) is a major zoonotic pathogen that causes hemorrhagic fever with renal syndrome (HFRS) in Asia, especially in China. Shaanxi province, which is located in northwest of China, is one of the areas in China most severely afflicted with HFRS epidemics annually. This study aims to...

  • Real-time quantitative analysis of the influence of blue light on citrinin biosynthetic gene cluster expression in Monascus. Wang, Changlu; Yang, Hua; Chen, Mianhua; Wang, Yurong; Li, Fengjuan; Luo, Cheng; Zhao, Shuyi; He, Dong // Biotechnology Letters;Sep2012, Vol. 34 Issue 9, p1745 

    When Monascus MX was grown under blue light instead of in the dark, citrinin production increased from 478 mg l to 698 mg l. To explain this, the expression of the pksCT gene, which encodes citrinin polyketide synthase, and of 5 ORFs around it, were monitored. Blue light enhanced citrinin...


Read the Article


Sorry, but this item is not currently available from your library.

Try another library?
Sign out of this library

Other Topics