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)
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 re...
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ftchinacasciocas:oai:ir.qdio.ac.cn:337002/11831 2023-05-15T18:15:43+02:00 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 Li Sun 2011-02-01 http://ir.qdio.ac.cn/handle/337002/11832 https://doi.org/10.1016/j.fsi.2010.12.028 英语 eng FISH & SHELLFISH IMMUNOLOGY 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),FISH & SHELLFISH IMMUNOLOGY,2011,30(2):720-728 http://ir.qdio.ac.cn/handle/337002/11832 doi:10.1016/j.fsi.2010.12.028 Quantitative Real Time Pcr Reference Gene Scophthalmus Maximus Housekeeping Gene Normalization Fisheries Immunology Marine & Freshwater Biology Veterinary Sciences Article 期刊论文 2011 ftchinacasciocas https://doi.org/10.1016/j.fsi.2010.12.028 2022-06-27T05:34:13Z 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 ... Article in Journal/Newspaper Scophthalmus maximus Turbot Institute of Oceanology, Chinese Academy of Sciences: IOCAS-IR Fish & Shellfish Immunology 30 2 720 728 |
institution |
Open Polar |
collection |
Institute of Oceanology, Chinese Academy of Sciences: IOCAS-IR |
op_collection_id |
ftchinacasciocas |
language |
English |
topic |
Quantitative Real Time Pcr Reference Gene Scophthalmus Maximus Housekeeping Gene Normalization Fisheries Immunology Marine & Freshwater Biology Veterinary Sciences |
spellingShingle |
Quantitative Real Time Pcr Reference Gene Scophthalmus Maximus Housekeeping Gene Normalization Fisheries Immunology Marine & Freshwater Biology Veterinary Sciences 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) |
topic_facet |
Quantitative Real Time Pcr Reference Gene Scophthalmus Maximus Housekeeping Gene Normalization Fisheries Immunology Marine & Freshwater Biology Veterinary Sciences |
description |
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 ... |
format |
Article in Journal/Newspaper |
author |
Wei Dang Li Sun |
author_facet |
Wei Dang Li Sun |
author_sort |
Wei Dang |
title |
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) |
title_short |
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) |
title_full |
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) |
title_fullStr |
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) |
title_full_unstemmed |
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) |
title_sort |
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) |
publishDate |
2011 |
url |
http://ir.qdio.ac.cn/handle/337002/11832 https://doi.org/10.1016/j.fsi.2010.12.028 |
genre |
Scophthalmus maximus Turbot |
genre_facet |
Scophthalmus maximus Turbot |
op_relation |
FISH & SHELLFISH IMMUNOLOGY 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),FISH & SHELLFISH IMMUNOLOGY,2011,30(2):720-728 http://ir.qdio.ac.cn/handle/337002/11832 doi:10.1016/j.fsi.2010.12.028 |
op_doi |
https://doi.org/10.1016/j.fsi.2010.12.028 |
container_title |
Fish & Shellfish Immunology |
container_volume |
30 |
container_issue |
2 |
container_start_page |
720 |
op_container_end_page |
728 |
_version_ |
1766188916377911296 |