不同饲料硒水平饲养大鼠的多组织内参基因筛选  

Screening for reference genes in various tissues of rats fed at different dietary selenium concentrations

作　　者：梁雄顺 莫俊銮[1] 龚春梅[1] 徐远飞[1] 刘小立[1] 杨晓光 周继昌[1] Liang Xiongshun;Mo Junluan;Gong Chunmei;Xu Yuanfei;Liu Xiaoli;Yang Xiaoguang;Zhou Jichang(Shenzhen Center for Chronic Disease Control,Shenzhen 518020,China;National Institute for Nutrition and Health,Chinese Center for Disease Control and Prevention,Beijing 100050,China)

机构地区：[1]深圳市慢性病防治中心,深圳518020 [2]中国疾病预防控制中心营养与健康所,北京100050

出　　处：《卫生研究》2019年第1期89-93,98,共6页Journal of Hygiene Research

基　　金：国家自然科学基金(No.81172669;81372993);深圳市医疗卫生三名工程(No.营养SZSM201611017)

摘　　要：目的筛选不同饲料硒(selenium,Se)水平饲喂大鼠时,不同组织中稳定表达的内参基因(reference genes,RGs)。方法 24只断乳雄性SD大鼠在缺Se饲养5周后,随机均分为4组,分别以Se含量<0.01、0.25、3、5 mg/kg饲料饲喂4周后处死,取肝、睾丸、骨骼肌、脂肪组织等样品待检。以荧光定量PCR检测Actb、Atp5f1、B2m、Gapdh、Gusb、Hprt、Pgk1、Ppia、Rplp2、Rps18、Tbp、Ywhaz等12个候选内参基因的mRNA水平,以geNorm、NormFinder、BestKeeper、Delta CT和RefFinder等方法对其表达稳定性进行评价。结果各组织中稳定性排名前4的内参基因是:肝中Ppia>Atp5f1>Rplp2>Hprt;睾丸中Ywhaz>Atp5f1>Rplp2>Ppia;骨骼肌中Tbp>Ppia>B2m>Rps18;脂肪组织中Hprt>Tbp>Atp5f1>Pgk1;综合4种组织,则Rps18>Hprt>Rplp2>Atp5f1。结论分析不同饲料Se水平饲养大鼠的目标基因表达水平时,应根据组织类型选择适宜的内参基因。OBJECTIVE To screen for the most stable reference genes (RGs) in various tissues of rats fed at different dietary concentrations of selenium (Se). METHODS Twenty-four weaning male SD rats were fed Se deficient diet for 5 weeks, and then randomly divided into 4 groups for<0.01,0.25,3 and 5 mg Se/kg diet feeding, respectively.After 4 weeks,animals were sacrificed for sample collection of liver,testis, muscle and fat tissue.Twelve candidate RGs of Actb,AtpSfl,B2m,Gapdh,Gusb,Hprt, Pgkl,Ppia,Rplp2,Rpsl8,Tbp and Ywhaz were tested for their quantitative cycle numbers of mRNA abundances with the quantitative PCR method .The stabilities of the candidate RGs were evaluated by the arithmetic packages of geNorm,NormFinder, BestKeeper,Delta CT and RefFinder.RESULTS The top 4 most stable RGs were Ppia >Atp5fl >Rplp2 >Hprt in liver;Ywhaz >AtpSfl >Rplp2>Ppia in testis;Tbp >Ppia > B2m >Rpsl8 in muscle;Hprt>Tbp >AtpSfl>Pgkl in fat tissue;and Rpsl8>Hprt>Rplp2>AtpSfl when all the 4 tissues combined for analysis.CONCLUSION To analyze the expressions of the target genes in rats fed different concentrations of dietary Se,the best RGs should be selected depending on the tissue types.

关 键 词：内参基因 定量PCR 饲料硒 大鼠 多组织 

分 类 号：Q581[生物学—生物化学] Q28