Evaluation of new reference genes in papaya for accurate transcript normalization under different experimental conditions.

Real-time reverse transcription PCR (RT-qPCR) is a preferred method for rapid and accurate quantification of gene expression studies. Appropriate application of RT-qPCR requires accurate normalization though the use of reference genes. As no single reference gene is universally suitable for all expe...

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Autores principales:	Xiaoyang Zhu, Xueping Li, Weixin Chen, Jianye Chen, Wangjin Lu, Lei Chen, Danwen Fu
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/0ac781949d4e45bc858cbf8ddc7e6ca9
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spelling	oai:doaj.org-article:0ac781949d4e45bc858cbf8ddc7e6ca92021-11-18T07:06:49ZEvaluation of new reference genes in papaya for accurate transcript normalization under different experimental conditions.1932-620310.1371/journal.pone.0044405https://doaj.org/article/0ac781949d4e45bc858cbf8ddc7e6ca92012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22952972/?tool=EBIhttps://doaj.org/toc/1932-6203Real-time reverse transcription PCR (RT-qPCR) is a preferred method for rapid and accurate quantification of gene expression studies. Appropriate application of RT-qPCR requires accurate normalization though the use of reference genes. As no single reference gene is universally suitable for all experiments, thus reference gene(s) validation under different experimental conditions is crucial for RT-qPCR analysis. To date, only a few studies on reference genes have been done in other plants but none in papaya. In the present work, we selected 21 candidate reference genes, and evaluated their expression stability in 246 papaya fruit samples using three algorithms, geNorm, NormFinder and RefFinder. The samples consisted of 13 sets collected under different experimental conditions, including various tissues, different storage temperatures, different cultivars, developmental stages, postharvest ripening, modified atmosphere packaging, 1-methylcyclopropene (1-MCP) treatment, hot water treatment, biotic stress and hormone treatment. Our results demonstrated that expression stability varied greatly between reference genes and that different suitable reference gene(s) or combination of reference genes for normalization should be validated according to the experimental conditions. In general, the internal reference genes EIF (Eukaryotic initiation factor 4A), TBP1 (TATA binding protein 1) and TBP2 (TATA binding protein 2) genes had a good performance under most experimental conditions, whereas the most widely present used reference genes, ACTIN (Actin 2), 18S rRNA (18S ribosomal RNA) and GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) were not suitable in many experimental conditions. In addition, two commonly used programs, geNorm and Normfinder, were proved sufficient for the validation. This work provides the first systematic analysis for the selection of superior reference genes for accurate transcript normalization in papaya under different experimental conditions.Xiaoyang ZhuXueping LiWeixin ChenJianye ChenWangjin LuLei ChenDanwen FuPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 8, p e44405 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Xiaoyang Zhu Xueping Li Weixin Chen Jianye Chen Wangjin Lu Lei Chen Danwen Fu Evaluation of new reference genes in papaya for accurate transcript normalization under different experimental conditions.
description	Real-time reverse transcription PCR (RT-qPCR) is a preferred method for rapid and accurate quantification of gene expression studies. Appropriate application of RT-qPCR requires accurate normalization though the use of reference genes. As no single reference gene is universally suitable for all experiments, thus reference gene(s) validation under different experimental conditions is crucial for RT-qPCR analysis. To date, only a few studies on reference genes have been done in other plants but none in papaya. In the present work, we selected 21 candidate reference genes, and evaluated their expression stability in 246 papaya fruit samples using three algorithms, geNorm, NormFinder and RefFinder. The samples consisted of 13 sets collected under different experimental conditions, including various tissues, different storage temperatures, different cultivars, developmental stages, postharvest ripening, modified atmosphere packaging, 1-methylcyclopropene (1-MCP) treatment, hot water treatment, biotic stress and hormone treatment. Our results demonstrated that expression stability varied greatly between reference genes and that different suitable reference gene(s) or combination of reference genes for normalization should be validated according to the experimental conditions. In general, the internal reference genes EIF (Eukaryotic initiation factor 4A), TBP1 (TATA binding protein 1) and TBP2 (TATA binding protein 2) genes had a good performance under most experimental conditions, whereas the most widely present used reference genes, ACTIN (Actin 2), 18S rRNA (18S ribosomal RNA) and GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) were not suitable in many experimental conditions. In addition, two commonly used programs, geNorm and Normfinder, were proved sufficient for the validation. This work provides the first systematic analysis for the selection of superior reference genes for accurate transcript normalization in papaya under different experimental conditions.
format	article
author	Xiaoyang Zhu Xueping Li Weixin Chen Jianye Chen Wangjin Lu Lei Chen Danwen Fu
author_facet	Xiaoyang Zhu Xueping Li Weixin Chen Jianye Chen Wangjin Lu Lei Chen Danwen Fu
author_sort	Xiaoyang Zhu
title	Evaluation of new reference genes in papaya for accurate transcript normalization under different experimental conditions.
title_short	Evaluation of new reference genes in papaya for accurate transcript normalization under different experimental conditions.
title_full	Evaluation of new reference genes in papaya for accurate transcript normalization under different experimental conditions.
title_fullStr	Evaluation of new reference genes in papaya for accurate transcript normalization under different experimental conditions.
title_full_unstemmed	Evaluation of new reference genes in papaya for accurate transcript normalization under different experimental conditions.
title_sort	evaluation of new reference genes in papaya for accurate transcript normalization under different experimental conditions.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/0ac781949d4e45bc858cbf8ddc7e6ca9
work_keys_str_mv	AT xiaoyangzhu evaluationofnewreferencegenesinpapayaforaccuratetranscriptnormalizationunderdifferentexperimentalconditions AT xuepingli evaluationofnewreferencegenesinpapayaforaccuratetranscriptnormalizationunderdifferentexperimentalconditions AT weixinchen evaluationofnewreferencegenesinpapayaforaccuratetranscriptnormalizationunderdifferentexperimentalconditions AT jianyechen evaluationofnewreferencegenesinpapayaforaccuratetranscriptnormalizationunderdifferentexperimentalconditions AT wangjinlu evaluationofnewreferencegenesinpapayaforaccuratetranscriptnormalizationunderdifferentexperimentalconditions AT leichen evaluationofnewreferencegenesinpapayaforaccuratetranscriptnormalizationunderdifferentexperimentalconditions AT danwenfu evaluationofnewreferencegenesinpapayaforaccuratetranscriptnormalizationunderdifferentexperimentalconditions
_version_	1718423895973822464

Evaluation of new reference genes in papaya for accurate transcript normalization under different experimental conditions.

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