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Selection of Suitable Endogenous Reference Genes for Relative Copy Number Detection in Sugarcane

Transgene copy number has a great impact on the expression level and stability of exogenous gene in transgenic plants. Proper selection of endogenous reference genes is necessary for detection of genetic components in genetically modification (GM) crops by quantitative real-time PCR (qPCR) or by qua...

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Autores principales: Xue, Bantong, Guo, Jinlong, Que, Youxiong, Fu, Zhiwei, Wu, Luguang, Xu, Liping
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Molecular Diversity Preservation International (MDPI) 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4057763/
https://www.ncbi.nlm.nih.gov/pubmed/24857916
http://dx.doi.org/10.3390/ijms15058846
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author Xue, Bantong
Guo, Jinlong
Que, Youxiong
Fu, Zhiwei
Wu, Luguang
Xu, Liping
author_facet Xue, Bantong
Guo, Jinlong
Que, Youxiong
Fu, Zhiwei
Wu, Luguang
Xu, Liping
author_sort Xue, Bantong
collection PubMed
description Transgene copy number has a great impact on the expression level and stability of exogenous gene in transgenic plants. Proper selection of endogenous reference genes is necessary for detection of genetic components in genetically modification (GM) crops by quantitative real-time PCR (qPCR) or by qualitative PCR approach, especially in sugarcane with polyploid and aneuploid genomic structure. qPCR technique has been widely accepted as an accurate, time-saving method on determination of copy numbers in transgenic plants and on detection of genetically modified plants to meet the regulatory and legislative requirement. In this study, to find a suitable endogenous reference gene and its real-time PCR assay for sugarcane (Saccharum spp. hybrids) DNA content quantification, we evaluated a set of potential “single copy” genes including P4H, APRT, ENOL, CYC, TST and PRR, through qualitative PCR and absolute quantitative PCR. Based on copy number comparisons among different sugarcane genotypes, including five S. officinarum, one S. spontaneum and two S. spp. hybrids, these endogenous genes fell into three groups: ENOL-3—high copy number group, TST-1 and PRR-1—medium copy number group, P4H-1, APRT-2 and CYC-2—low copy number group. Among these tested genes, P4H, APRT and CYC were the most stable, while ENOL and TST were the least stable across different sugarcane genotypes. Therefore, three primer pairs of P4H-3, APRT-2 and CYC-2 were then selected as the suitable reference gene primer pairs for sugarcane. The test of multi-target reference genes revealed that the APRT gene was a specific amplicon, suggesting this gene is the most suitable to be used as an endogenous reference target for sugarcane DNA content quantification. These results should be helpful for establishing accurate and reliable qualitative and quantitative PCR analysis of GM sugarcane.
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spelling pubmed-40577632014-06-16 Selection of Suitable Endogenous Reference Genes for Relative Copy Number Detection in Sugarcane Xue, Bantong Guo, Jinlong Que, Youxiong Fu, Zhiwei Wu, Luguang Xu, Liping Int J Mol Sci Article Transgene copy number has a great impact on the expression level and stability of exogenous gene in transgenic plants. Proper selection of endogenous reference genes is necessary for detection of genetic components in genetically modification (GM) crops by quantitative real-time PCR (qPCR) or by qualitative PCR approach, especially in sugarcane with polyploid and aneuploid genomic structure. qPCR technique has been widely accepted as an accurate, time-saving method on determination of copy numbers in transgenic plants and on detection of genetically modified plants to meet the regulatory and legislative requirement. In this study, to find a suitable endogenous reference gene and its real-time PCR assay for sugarcane (Saccharum spp. hybrids) DNA content quantification, we evaluated a set of potential “single copy” genes including P4H, APRT, ENOL, CYC, TST and PRR, through qualitative PCR and absolute quantitative PCR. Based on copy number comparisons among different sugarcane genotypes, including five S. officinarum, one S. spontaneum and two S. spp. hybrids, these endogenous genes fell into three groups: ENOL-3—high copy number group, TST-1 and PRR-1—medium copy number group, P4H-1, APRT-2 and CYC-2—low copy number group. Among these tested genes, P4H, APRT and CYC were the most stable, while ENOL and TST were the least stable across different sugarcane genotypes. Therefore, three primer pairs of P4H-3, APRT-2 and CYC-2 were then selected as the suitable reference gene primer pairs for sugarcane. The test of multi-target reference genes revealed that the APRT gene was a specific amplicon, suggesting this gene is the most suitable to be used as an endogenous reference target for sugarcane DNA content quantification. These results should be helpful for establishing accurate and reliable qualitative and quantitative PCR analysis of GM sugarcane. Molecular Diversity Preservation International (MDPI) 2014-05-19 /pmc/articles/PMC4057763/ /pubmed/24857916 http://dx.doi.org/10.3390/ijms15058846 Text en © 2014 by the authors; licensee MDPI, Basel, Switzerland http://creativecommons.org/licenses/by/3.0/ This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
spellingShingle Article
Xue, Bantong
Guo, Jinlong
Que, Youxiong
Fu, Zhiwei
Wu, Luguang
Xu, Liping
Selection of Suitable Endogenous Reference Genes for Relative Copy Number Detection in Sugarcane
title Selection of Suitable Endogenous Reference Genes for Relative Copy Number Detection in Sugarcane
title_full Selection of Suitable Endogenous Reference Genes for Relative Copy Number Detection in Sugarcane
title_fullStr Selection of Suitable Endogenous Reference Genes for Relative Copy Number Detection in Sugarcane
title_full_unstemmed Selection of Suitable Endogenous Reference Genes for Relative Copy Number Detection in Sugarcane
title_short Selection of Suitable Endogenous Reference Genes for Relative Copy Number Detection in Sugarcane
title_sort selection of suitable endogenous reference genes for relative copy number detection in sugarcane
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4057763/
https://www.ncbi.nlm.nih.gov/pubmed/24857916
http://dx.doi.org/10.3390/ijms15058846
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