Assessment of internal controls for data normalization of gene expression after different bacterial stimulation by quantitative real-time PCR in golden pompano Trachinotus blochii 被引量：1

Assessment of internal controls for data normalization of gene expression after different bacterial stimulation by quantitative real-time PCR in golden pompano Trachinotus blochii

下载PDF

导出

摘要 Trachinotus blochii is one of the important commercial fish species.In this study,we aim to confirm the reliability reference genes in T.blochii during different bacterial challenge through quantitative real-time PCR(qRT-PCR).The expression of the seven selected genes in four immune organs(i.e.,spleen,kidney,intestine,and gill)stimulated with Vibrio harveyi,Edwardsiella tarda,and Streptococcus agalactiae were determined by qRT-PCR.The PCR data was analyzed using the geNorm and NormFinder algorithms.The results showed the selection of the internal controls should be tissue specific when studying gene expression in response to bacterial stimulation.After 48 h of stimulation with V.harveyi,geNorm ranked EF1 A/Actin,18 S rRNA/B2M,UBCE/B2M,and 18 S rRNA/B2M,as the most stably expressed genes in spleen,kidney,intestine,and gill,respectively.After 48 h of stimulation with E.tarda,geNorm ranked 18 S rRNA/EF1 A,18 S rRNA/B2M,B2M/RPL13,and 18 S rRNA/EF1 A,as the most stably expressed genes in spleen,kidney,intestine,and gill,respectively.After 48 h of stimulation with S.agalactiae,18 S rRNA/EF1 A,18 S rRNA/B2 M,B2 M/Actin,and 18 S rRNA/B2M were ranked as the most stably expressed genes in spleen,kidney,intestine,and gill,respectively.Compared to the results analyzed by geNorm,reference genes received similar rankings when using NormFinder software.The results showed that the reference genes appeared to be not only tissue specific,but also specific to the infecting species of bacteria.If one gene is preferred when T.blochii were infected by bacteria,18 S rRNA,B2M,B2M,18 S rRNA may be used in spleen,kidney,intestine,and gill,respectively. Trachinotus blochii is one of the important commercial fish species.In this study,we aim to confirm the reliability reference genes in T.blochii during different bacterial challenge through quantitative real-time PCR(qRT-PCR).The expression of the seven selected genes in four immune organs(i.e.,spleen,kidney,intestine,and gill) stimulated with Vibrio harveyi,Edwardsiella tarda,and Streptococcus agalactiae were determined by qRT-PCR.The PCR data was analyzed using the geNorm and NormFinder algorithms.The results showed the selection of the internal controls should be tissue specific when studying gene expression in response to bacterial stimulation.After 48 h of stimulation with V.harveyi,geNorm ranked EF1 A/Actin,18 S rRNA/B2M,UBCE/B2M,and 18 S rRNA/B2M,as the most stably expressed genes in spleen,kidney,intestine,and gill,respectively.After 48 h of stimulation with E.tarda,geNorm ranked 18 S rRNA/EF1 A,18 S rRNA/B2M,B2M/RPL13,and 18 S rRNA/EF1 A,as the most stably expressed genes in spleen,kidney,intestine,and gill,respectively.After 48 h of stimulation with S.agalactiae,18 S rRNA/EF1 A,18 S rRNA/B2 M,B2 M/Actin,and 18 S rRNA/B2M were ranked as the most stably expressed genes in spleen,kidney,intestine,and gill,respectively.Compared to the results analyzed by geNorm,reference genes received similar rankings when using NormFinder software.The results showed that the reference genes appeared to be not only tissue specific,but also specific to the infecting species of bacteria.If one gene is preferred when T.blochii were infected by bacteria,18 S rRNA,B2M,B2M,18 S rRNA may be used in spleen,kidney,intestine,and gill,respectively.

作者 CHEN Xiaojuan ZHANG Xiaoqi SUN Yun TU Zhigang CAO Zhenjie WANG Shifeng ZHOU Yongcan

机构地区 State Key Laboratory of Marine Resource Utilization in South China Sea Hainan Academy of Ocean and Fisheries Sciences

出处《Journal of Oceanology and Limnology》 SCIE CAS CSCD 2020年第2期480-489,共10页 海洋湖沼学报（英文）

基金 Supported by the National Natural Science Foundation of China(No.41666006,31702379) the Nanhai Famous Youth Project

关键词 Trachinotus blochii housekeeping gene expression stability reference gene Trachinotus blochii housekeeping gene expression stability reference gene

分类号 S917.4 [农业科学—水产科学]

引文网络
相关文献

参考文献1

1李迪,吴萍,何美凤,李伟,肖调义,褚武英.qRT-PCR分析鳜鱼内参基因的筛选[J].生命科学研究,2016,20(3):214-217. 被引量：15

二级参考文献21

1王修启,谭会泽,苏海林,宋予震,冯定远.内标18S rRNA和-βactin mRNA比较:肉鸡十二指肠SGLT1 mRNA的发育规律[J].华北农学报,2006,21(3):112-116. 被引量：3
2张艳君,朱志峰,陆融,徐琼,石琳熙,简序,刘俊燕,姚智.基因表达转录分析中内参基因的选择[J].生物化学与生物物理进展,2007,34(5):546-550. 被引量：79
3HEID C A, STEVENS J, LIVAK K J, et aL Real time quanti- tative PCR[J]. Genome Research, 1996, 6(10): 986-994.
4HALLER F, KULLE B, SCHWAGER S, et al. Equivalence test in quantitative reverse transcription polymerase chain reaction: confirmation of reference genes suitable for normalization [J]. Analytical Biochemistry, 2004, 12(1): 1-9.
5RANSBOTYN V, REUSCH T B H. Housekeeping gene selec- tion for quantitative real-time PCR assays in the seagrass Zostera marina subjected to heat stress[J]. Limnol and Oceanog- raphy: Methods, 2006, 4(10): 367-373.
6YOO W G, KIM T I, LI S Y, et al. Reference genes for quan- titative analysis on Clonorchis sinensis gene expression by re- al-time PCR[J]. Parasitology Research, 2009, 104(2): 321-328.
7I,IU D W, CHEN S T, LIU H P. Choice of endogenous control for gene expression in nonsmall cell lung cancer[J]. European Res- piratory Journal, 2005, 26(6): 1002-1008.
8SELVEY S, THOMPSON E W, NI*I'HAEI M. :-actin an un- suitable internal control for RT-PCR[J: Molecular and Cell Probes, 2001, 15(5): 307-311.
9BUSTIN S A. Absolute quantification of mRNA using real- time reverse transcription polymerase chain reaction assays[J]. Journal of Molecular Endocrinology, 2000, 25(2):169-193.
10ZHU X, LI Y L, CHEN D X, et al. Selection of reference genes for microRNA quantitative expression analysis in Chinese perch, Siniperca chuatsi[J]. International Journal of Molecular Sciences, 2015, 16(4): 8310-8323.