Determination of internal controls for quantitative real time RT-PCR analysis of the effect of Edwardsiella tarda infection on gene expression in turbot (Scophthalmus maximus)

doi:10.1016/j.fsi.2010.12.028

CORC > 海洋研究所 > 中国科学院海洋研究所 > 实验海洋生物学重点实验室

	Determination of internal controls for quantitative real time RT-PCR analysis of the effect of Edwardsiella tarda infection on gene expression in turbot (Scophthalmus maximus)
	Wei Dang 1,2; Li Sun 1
刊名	FISH & SHELLFISH IMMUNOLOGY
	2011-02-01
卷号	30 期号:2 页码:720-728
关键词	Quantitative Real Time Pcr Reference Gene Scophthalmus Maximus Housekeeping Gene Normalization
ISSN号	1050-4648
DOI	10.1016/j.fsi.2010.12.028
文献子类	Article
英文摘要	In recent years, quantitative real time reverse transcriptase-PCR (qRT-PCR) has been used frequently in the study of gene expression in turbot (Scophthalmus maximus) in relation to bacterial infection. However, no investigations on appropriate qRT-PCR reference genes have been documented. In this report, we determined the potential of eight housekeeping genes, i.e. beta-actin (ACTB), ribosomal protein L17 (RPL17), alpha-tubulin (TUBA), elongation factor-1-alpha(EF1A), beta-2-Microglobulin (B2M), RNA polymerase II subunit D (RPSD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and 185 ribosomal RNA (18S rRNA), as internal standards for qRT-PCR analysis of gene expression in turbot as a function of bacterial infection. For this purpose, the expression of the eight housekeeping genes in seven turbot tissues was determined by qRT-PCR before and after bacterial challenge, and the data were analyzed with the geNorm and NormFinder algorisms. The results showed that the expression of all the examined genes exhibited tissue-dependent variations both before and after bacterial challenge. Before bacterial challenge, geNorm and NormFinder identified RPSD as the gene that showed least tissue specific expression. At 12 h post-bacterial infection, geNorm ranked ACTB/GAPDH, 18S rRNA/ACTB, ACTB/GAPDH, 18S rRNA/ACTB, RPL17/TUBA, RPSD/GAPDH, and RPSD/B2M, respectively, as the most stably expressed genes in liver, spleen, kidney, gill, heart, muscle, and brain. Comparable ranking orders were produced by NormFinder. Similar results were obtained at 24 h post-bacterial infection. Taken together, these results indicate that RPSD is the most stable gene across tissue types under normal physiological conditions and that, during bacterial infection, ACTS might be used as an internal standard for the normalization of gene expression in immune relevant organs; however, no single gene or single pair of genes in the examined set of housekeeping genes can serve as a universal reference across all tissue types under the condition of bacterial infection. (C) 2011 Elsevier Ltd. All rights reserved.; In recent years, quantitative real time reverse transcriptase-PCR (qRT-PCR) has been used frequently in the study of gene expression in turbot (Scophthalmus maximus) in relation to bacterial infection. However, no investigations on appropriate qRT-PCR reference genes have been documented. In this report, we determined the potential of eight housekeeping genes, i.e. beta-actin (ACTB), ribosomal protein L17 (RPL17), alpha-tubulin (TUBA), elongation factor-1-alpha(EF1A), beta-2-Microglobulin (B2M), RNA polymerase II subunit D (RPSD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and 185 ribosomal RNA (18S rRNA), as internal standards for qRT-PCR analysis of gene expression in turbot as a function of bacterial infection. For this purpose, the expression of the eight housekeeping genes in seven turbot tissues was determined by qRT-PCR before and after bacterial challenge, and the data were analyzed with the geNorm and NormFinder algorisms. The results showed that the expression of all the examined genes exhibited tissue-dependent variations both before and after bacterial challenge. Before bacterial challenge, geNorm and NormFinder identified RPSD as the gene that showed least tissue specific expression. At 12 h post-bacterial infection, geNorm ranked ACTB/GAPDH, 18S rRNA/ACTB, ACTB/GAPDH, 18S rRNA/ACTB, RPL17/TUBA, RPSD/GAPDH, and RPSD/B2M, respectively, as the most stably expressed genes in liver, spleen, kidney, gill, heart, muscle, and brain. Comparable ranking orders were produced by NormFinder. Similar results were obtained at 24 h post-bacterial infection. Taken together, these results indicate that RPSD is the most stable gene across tissue types under normal physiological conditions and that, during bacterial infection, ACTS might be used as an internal standard for the normalization of gene expression in immune relevant organs; however, no single gene or single pair of genes in the examined set of housekeeping genes can serve as a universal reference across all tissue types under the condition of bacterial infection. (C) 2011 Elsevier Ltd. All rights reserved.
学科主题	Fisheries ; Immunology ; Marine & Freshwater Biology ; Veterinary Sciences
URL标识	查看原文
语种	英语
WOS记录号	WOS:000288305300034
公开日期	2012-07-03
内容类型	期刊论文
源URL	[http://ir.qdio.ac.cn/handle/337002/11832]
专题	海洋研究所_实验海洋生物学重点实验室
作者单位	1.Chinese Acad Sci, Inst Oceanol, Key Lab Expt Marine Biol, Qingdao 266071, Peoples R China 2.Chinese Acad Sci, Grad Univ, Beijing 100049, Peoples R China
推荐引用方式 GB/T 7714	Wei Dang,Li Sun. Determination of internal controls for quantitative real time RT-PCR analysis of the effect of Edwardsiella tarda infection on gene expression in turbot (Scophthalmus maximus)[J]. FISH & SHELLFISH IMMUNOLOGY,2011,30(2):720-728.
APA	Wei Dang,&Li Sun.(2011).Determination of internal controls for quantitative real time RT-PCR analysis of the effect of Edwardsiella tarda infection on gene expression in turbot (Scophthalmus maximus).FISH & SHELLFISH IMMUNOLOGY,30(2),720-728.
MLA	Wei Dang,et al."Determination of internal controls for quantitative real time RT-PCR analysis of the effect of Edwardsiella tarda infection on gene expression in turbot (Scophthalmus maximus)".FISH & SHELLFISH IMMUNOLOGY 30.2(2011):720-728.