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Selection and validation of a set of reliable reference genes for quantitative sod gene expression analysis in C. elegans
BACKGROUND: In the nematode Caenorhabditis elegans the conserved Ins/IGF-1 signaling pathway regulates many biological processes including life span, stress response, dauer diapause and metabolism. Detection of differentially expressed genes may contribute to a better understanding of the mechanism...
Autores principales: | , , , , |
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Formato: | Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2008
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2254638/ https://www.ncbi.nlm.nih.gov/pubmed/18211699 http://dx.doi.org/10.1186/1471-2199-9-9 |
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author | Hoogewijs, David Houthoofd, Koen Matthijssens, Filip Vandesompele, Jo Vanfleteren, Jacques R |
author_facet | Hoogewijs, David Houthoofd, Koen Matthijssens, Filip Vandesompele, Jo Vanfleteren, Jacques R |
author_sort | Hoogewijs, David |
collection | PubMed |
description | BACKGROUND: In the nematode Caenorhabditis elegans the conserved Ins/IGF-1 signaling pathway regulates many biological processes including life span, stress response, dauer diapause and metabolism. Detection of differentially expressed genes may contribute to a better understanding of the mechanism by which the Ins/IGF-1 signaling pathway regulates these processes. Appropriate normalization is an essential prerequisite for obtaining accurate and reproducible quantification of gene expression levels. The aim of this study was to establish a reliable set of reference genes for gene expression analysis in C. elegans. RESULTS: Real-time quantitative PCR was used to evaluate the expression stability of 12 candidate reference genes (act-1, ama-1, cdc-42, csq-1, eif-3.C, mdh-1, gpd-2, pmp-3, tba-1, Y45F10D.4, rgs-6 and unc-16) in wild-type, three Ins/IGF-1 pathway mutants, dauers and L3 stage larvae. After geNorm analysis, cdc-42, pmp-3 and Y45F10D.4 showed the most stable expression pattern and were used to normalize 5 sod expression levels. Significant differences in mRNA levels were observed for sod-1 and sod-3 in daf-2 relative to wild-type animals, whereas in dauers sod-1, sod-3, sod-4 and sod-5 are differentially expressed relative to third stage larvae. CONCLUSION: Our findings emphasize the importance of accurate normalization using stably expressed reference genes. The methodology used in this study is generally applicable to reliably quantify gene expression levels in the nematode C. elegans using quantitative PCR. |
format | Text |
id | pubmed-2254638 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2008 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-22546382008-02-27 Selection and validation of a set of reliable reference genes for quantitative sod gene expression analysis in C. elegans Hoogewijs, David Houthoofd, Koen Matthijssens, Filip Vandesompele, Jo Vanfleteren, Jacques R BMC Mol Biol Research Article BACKGROUND: In the nematode Caenorhabditis elegans the conserved Ins/IGF-1 signaling pathway regulates many biological processes including life span, stress response, dauer diapause and metabolism. Detection of differentially expressed genes may contribute to a better understanding of the mechanism by which the Ins/IGF-1 signaling pathway regulates these processes. Appropriate normalization is an essential prerequisite for obtaining accurate and reproducible quantification of gene expression levels. The aim of this study was to establish a reliable set of reference genes for gene expression analysis in C. elegans. RESULTS: Real-time quantitative PCR was used to evaluate the expression stability of 12 candidate reference genes (act-1, ama-1, cdc-42, csq-1, eif-3.C, mdh-1, gpd-2, pmp-3, tba-1, Y45F10D.4, rgs-6 and unc-16) in wild-type, three Ins/IGF-1 pathway mutants, dauers and L3 stage larvae. After geNorm analysis, cdc-42, pmp-3 and Y45F10D.4 showed the most stable expression pattern and were used to normalize 5 sod expression levels. Significant differences in mRNA levels were observed for sod-1 and sod-3 in daf-2 relative to wild-type animals, whereas in dauers sod-1, sod-3, sod-4 and sod-5 are differentially expressed relative to third stage larvae. CONCLUSION: Our findings emphasize the importance of accurate normalization using stably expressed reference genes. The methodology used in this study is generally applicable to reliably quantify gene expression levels in the nematode C. elegans using quantitative PCR. BioMed Central 2008-01-22 /pmc/articles/PMC2254638/ /pubmed/18211699 http://dx.doi.org/10.1186/1471-2199-9-9 Text en Copyright © 2008 Hoogewijs et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( (http://creativecommons.org/licenses/by/2.0) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Article Hoogewijs, David Houthoofd, Koen Matthijssens, Filip Vandesompele, Jo Vanfleteren, Jacques R Selection and validation of a set of reliable reference genes for quantitative sod gene expression analysis in C. elegans |
title | Selection and validation of a set of reliable reference genes for quantitative sod gene expression analysis in C. elegans |
title_full | Selection and validation of a set of reliable reference genes for quantitative sod gene expression analysis in C. elegans |
title_fullStr | Selection and validation of a set of reliable reference genes for quantitative sod gene expression analysis in C. elegans |
title_full_unstemmed | Selection and validation of a set of reliable reference genes for quantitative sod gene expression analysis in C. elegans |
title_short | Selection and validation of a set of reliable reference genes for quantitative sod gene expression analysis in C. elegans |
title_sort | selection and validation of a set of reliable reference genes for quantitative sod gene expression analysis in c. elegans |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2254638/ https://www.ncbi.nlm.nih.gov/pubmed/18211699 http://dx.doi.org/10.1186/1471-2199-9-9 |
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