title: Identification and Characterization of Reference Genes for Normalizing Expression Data from Red Swamp Crawfish Procambarus clarkii.
abstract: qRT-PCR is a widely used technique for rapid and accurate quantification of gene expression data. The use of reference genes for normalization of the expression levels is crucial for accuracy. Several studies have shown that there is no perfect reference gene that is appropriate for use in all experimental conditions, and research on suitable reference genes in red swamp crawfish (Procambarus clarkii) is particularly scarce. In this study, eight commonly used crustacean reference genes were chosen from P. clarkii transcriptome data and investigated as potential candidates for normalization of qRT-PCR data. Expression of these genes under different experimental conditions was examined by qRT-PCR, and the stability of their expression was evaluated using three commonly used statistical algorithms, geNorm, NormFinder and BestKeeper. A final comprehensive ranking determined that EIF and 18S were the optimal reference genes for expression data from different tissues, while TBP and EIF were optimal for expression data from different ovarian developmental stages. To our knowledge, this is the first systematic analysis of reference genes for normalization of qRT-PCR data in P. clarkii. These results will facilitate more accurate and reliable expression studies of this and other crustacean species.
authors.affiliation: Key Laboratory of Freshwater Fishery Germplasm Resources, Ministry of Agriculture, Shanghai Ocean University, Shanghai 201306, China. hcjiang@163.com.
authors.forename: Hucheng
authors.initials: H
authors.lastname: Jiang
authors.affiliation: Key Laboratory of Freshwater Fishery Germplasm Resources, Ministry of Agriculture, Shanghai Ocean University, Shanghai 201306, China. zhaojunqian1216@163.com.
authors.forename: Zhaojun
authors.initials: Z
authors.lastname: Qian
authors.affiliation: Jiangsu Xuyi Riverred Crawfish Eco-Park Co., Ltd., Xuyi 211700, China. 15851705385@163.com.
authors.forename: Wei
authors.initials: W
authors.lastname: Lu
authors.affiliation: Jiangsu Key Laboratory for Eco-Agricultural Biotechnoology Around Hongze Lake, Huaiyin Normal University, Huaian 223300, China. huaiyu-ding@163.com.
authors.forename: Huaiyu
authors.initials: H
authors.lastname: Ding
authors.affiliation: Jiangsu Xuyi Riverred Crawfish Eco-Park Co., Ltd., Xuyi 211700, China. yuhongwei-yhw@163.com.
authors.forename: Hongwei
authors.initials: H
authors.lastname: Yu
authors.affiliation: Jiangsu Key Laboratory for Eco-Agricultural Biotechnoology Around Hongze Lake, Huaiyin Normal University, Huaian 223300, China. hytcwhh@hytc.edu.cn.
authors.forename: Hui
authors.initials: H
authors.lastname: Wang
authors.affiliation: Key Laboratory of Freshwater Fishery Germplasm Resources, Ministry of Agriculture, Shanghai Ocean University, Shanghai 201306, China. jlli2009@126.com.
authors.forename: Jiale
authors.initials: J
authors.lastname: Li
mesh_terms: D000818:Animals; D003400:Astacoidea / Q000235:genetics*; D020869:Gene Expression Profiling; D016133:Polymerase Chain Reaction / Q000379:methods; D020871:RNA Stability; D015203:Reproducibility of Results; D059467:Transcriptome
keywords: Procambarus clarkii; developmental stage; normalization; qRT-PCR; reference gene