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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 Dang1,2; Li Sun1
2011-02-01
发表期刊FISH & SHELLFISH IMMUNOLOGY
ISSN1050-4648
卷号30期号:2页码:720-728
文章类型Article
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.
关键词Quantitative Real Time Pcr Reference Gene Scophthalmus Maximus Housekeeping Gene Normalization
学科领域Fisheries ; Immunology ; Marine & Freshwater Biology ; Veterinary Sciences
DOI10.1016/j.fsi.2010.12.028
URL查看原文
收录类别SCI
语种英语
WOS记录号WOS:000288305300034
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被引频次:74[WOS]   [WOS记录]     [WOS相关记录]
文献类型期刊论文
条目标识符http://ir.qdio.ac.cn/handle/337002/11831
专题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
第一作者单位实验海洋生物学重点实验室
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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.
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