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Comprehensive Selection of Reference Genes for Gene Expression Normalization in Sugarcane by Real Time Quantitative RT-PCR

The increasingly used real time quantitative reverse transcription-PCR (qRT-PCR) method for gene expression analysis requires one or several reference gene(s) acting as normalization factor(s). In order to facilitate gene expression studies in sugarcane (Saccharum officinarum), a non-model plant wit...

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Autores principales: Ling, Hui, Wu, Qibin, Guo, Jinlong, Xu, Liping, Que, Youxiong
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4019594/
https://www.ncbi.nlm.nih.gov/pubmed/24823940
http://dx.doi.org/10.1371/journal.pone.0097469
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author Ling, Hui
Wu, Qibin
Guo, Jinlong
Xu, Liping
Que, Youxiong
author_facet Ling, Hui
Wu, Qibin
Guo, Jinlong
Xu, Liping
Que, Youxiong
author_sort Ling, Hui
collection PubMed
description The increasingly used real time quantitative reverse transcription-PCR (qRT-PCR) method for gene expression analysis requires one or several reference gene(s) acting as normalization factor(s). In order to facilitate gene expression studies in sugarcane (Saccharum officinarum), a non-model plant with limited genome information, the stability of 13 candidate reference genes was evaluated. The geNorm, NormFinder and deltaCt methods were used for selecting stably expressed internal controls across different tissues and under various experimental treatments. These results revealed that, among these 13 candidate reference genes, GAPDH, eEF-1a and eIF-4α were the most stable and suitable for use as normalization factors across all various experimental samples. In addition, APRT could be a candidate for examining the relationship between gene copy number and transcript levels in sugarcane tissue samples. According to the results evaluated by geNorm, combining CUL and eEF-1α in hormone treatment experiments; CAC and CUL in abiotic stress tests; GAPDH, eEF-1a and CUL in all treatment samples plus CAC, CUL, APRT and TIPS-41 in cultivar tissues as groups for normalization would lead to more accurate and reliable expression quantification in sugarcane. This is the first systematic validation of reference genes for quantification of transcript expression profiles in sugarcane. This study should provide useful information for selecting reference genes for more accurate quantification of gene expression in sugarcane and other plant species.
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spelling pubmed-40195942014-05-16 Comprehensive Selection of Reference Genes for Gene Expression Normalization in Sugarcane by Real Time Quantitative RT-PCR Ling, Hui Wu, Qibin Guo, Jinlong Xu, Liping Que, Youxiong PLoS One Research Article The increasingly used real time quantitative reverse transcription-PCR (qRT-PCR) method for gene expression analysis requires one or several reference gene(s) acting as normalization factor(s). In order to facilitate gene expression studies in sugarcane (Saccharum officinarum), a non-model plant with limited genome information, the stability of 13 candidate reference genes was evaluated. The geNorm, NormFinder and deltaCt methods were used for selecting stably expressed internal controls across different tissues and under various experimental treatments. These results revealed that, among these 13 candidate reference genes, GAPDH, eEF-1a and eIF-4α were the most stable and suitable for use as normalization factors across all various experimental samples. In addition, APRT could be a candidate for examining the relationship between gene copy number and transcript levels in sugarcane tissue samples. According to the results evaluated by geNorm, combining CUL and eEF-1α in hormone treatment experiments; CAC and CUL in abiotic stress tests; GAPDH, eEF-1a and CUL in all treatment samples plus CAC, CUL, APRT and TIPS-41 in cultivar tissues as groups for normalization would lead to more accurate and reliable expression quantification in sugarcane. This is the first systematic validation of reference genes for quantification of transcript expression profiles in sugarcane. This study should provide useful information for selecting reference genes for more accurate quantification of gene expression in sugarcane and other plant species. Public Library of Science 2014-05-13 /pmc/articles/PMC4019594/ /pubmed/24823940 http://dx.doi.org/10.1371/journal.pone.0097469 Text en © 2014 Ling et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Ling, Hui
Wu, Qibin
Guo, Jinlong
Xu, Liping
Que, Youxiong
Comprehensive Selection of Reference Genes for Gene Expression Normalization in Sugarcane by Real Time Quantitative RT-PCR
title Comprehensive Selection of Reference Genes for Gene Expression Normalization in Sugarcane by Real Time Quantitative RT-PCR
title_full Comprehensive Selection of Reference Genes for Gene Expression Normalization in Sugarcane by Real Time Quantitative RT-PCR
title_fullStr Comprehensive Selection of Reference Genes for Gene Expression Normalization in Sugarcane by Real Time Quantitative RT-PCR
title_full_unstemmed Comprehensive Selection of Reference Genes for Gene Expression Normalization in Sugarcane by Real Time Quantitative RT-PCR
title_short Comprehensive Selection of Reference Genes for Gene Expression Normalization in Sugarcane by Real Time Quantitative RT-PCR
title_sort comprehensive selection of reference genes for gene expression normalization in sugarcane by real time quantitative rt-pcr
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4019594/
https://www.ncbi.nlm.nih.gov/pubmed/24823940
http://dx.doi.org/10.1371/journal.pone.0097469
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