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Identification and Validation of Reference Genes for Quantitative Real-Time PCR Normalization and Its Applications in Lycium
Alternative TitlePLoS One
Zeng, Shaohua1; Liu, Yongliang2; Wu, Min1; Liu, Xiaomin1; Shen, Xiaofei2; Liu, Chunzhao3; Wang, Ying1,2
2014-05-08
Source PublicationPLOS ONE
ISSN1932-6203
Volume9Issue:5Pages:13
AbstractLycium barbarum and L. ruthenicum are extensively used as traditional Chinese medicinal plants. Next generation sequencing technology provides a powerful tool for analyzing transcriptomic profiles of gene expression in non-model species. Such gene expression can then be confirmed with quantitative real-time polymerase chain reaction (qRT-PCR). Therefore, use of systematically identified suitable reference genes is a prerequisite for obtaining reliable gene expression data. Here, we calculated the expression stability of 18 candidate reference genes across samples from different tissues and grown under salt stress using geNorm and NormFinder procedures. The geNorm-determined rank of reference genes was similar to those defined by NormFinder with some differences. Both procedures confirmed that the single most stable reference gene was ACNTIN1 for L. barbarum fruits, H2B1 for L. barbarum roots, and EF1 alpha for L. ruthenicum fruits. PGK3, H2B2, and PGK3 were identified as the best stable reference genes for salt-treated L. ruthenicum leaves, roots, and stems, respectively. H2B1 and GAPDH1+PGK1 for L. ruthenicum and SAMDC2+H2B1 for L. barbarum were the best single and/or combined reference genes across all samples. Finally, expression of salt-responsive gene NAC, fruit ripening candidate gene LrPG, and anthocyanin genes were investigated to confirm the validity of the selected reference genes. Suitable reference genes identified in this study provide a foundation for accurately assessing gene expression and further better understanding of novel gene function to elucidate molecular mechanisms behind particular biological/physiological processes in Lycium.; Lycium barbarum and L. ruthenicum are extensively used as traditional Chinese medicinal plants. Next generation sequencing technology provides a powerful tool for analyzing transcriptomic profiles of gene expression in non-model species. Such gene expression can then be confirmed with quantitative real-time polymerase chain reaction (qRT-PCR). Therefore, use of systematically identified suitable reference genes is a prerequisite for obtaining reliable gene expression data. Here, we calculated the expression stability of 18 candidate reference genes across samples from different tissues and grown under salt stress using geNorm and NormFinder procedures. The geNorm-determined rank of reference genes was similar to those defined by NormFinder with some differences. Both procedures confirmed that the single most stable reference gene was ACNTIN1 for L. barbarum fruits, H2B1 for L. barbarum roots, and EF1 alpha for L. ruthenicum fruits. PGK3, H2B2, and PGK3 were identified as the best stable reference genes for salt-treated L. ruthenicum leaves, roots, and stems, respectively. H2B1 and GAPDH1+PGK1 for L. ruthenicum and SAMDC2+H2B1 for L. barbarum were the best single and/or combined reference genes across all samples. Finally, expression of salt-responsive gene NAC, fruit ripening candidate gene LrPG, and anthocyanin genes were investigated to confirm the validity of the selected reference genes. Suitable reference genes identified in this study provide a foundation for accurately assessing gene expression and further better understanding of novel gene function to elucidate molecular mechanisms behind particular biological/physiological processes in Lycium.
KeywordTomato Fruit Rt-pcr Housekeeping Genes Abiotic Stress Polygalacturonase Gene Regulatory Regions Expression Selection Rna Transcription
SubtypeArticle
WOS HeadingsScience & Technology
DOI10.1371/journal.pone.0097039
URL查看原文
Indexed BySCI
Language英语
WOS KeywordTOMATO FRUIT ; RT-PCR ; HOUSEKEEPING GENES ; ABIOTIC STRESS ; POLYGALACTURONASE GENE ; REGULATORY REGIONS ; EXPRESSION ; SELECTION ; RNA ; TRANSCRIPTION
WOS Research AreaScience & Technology - Other Topics
WOS SubjectMultidisciplinary Sciences
WOS IDWOS:000338213300112
Citation statistics
Cited Times:19[WOS]   [WOS Record]     [Related Records in WOS]
Document Type期刊论文
Version出版稿
Identifierhttp://ir.ipe.ac.cn/handle/122111/11020
Collection研究所(批量导入)
Affiliation1.Chinese Acad Sci, South China Bot Garden, Key Lab Plant Resources Conservat & Sustainable U, Guangzhou, Guangdong, Peoples R China
2.Chinese Acad Sci, Wuhan Bot Garden, Key Lab Pant Germplasm Enhancement & Specialty Ag, Wuhan, Hubei, Peoples R China
3.Chinese Acad Sci, Inst Proc Engn, Natl Key Lab Biochem Engn, Beijing, Peoples R China
Recommended Citation
GB/T 7714
Zeng, Shaohua,Liu, Yongliang,Wu, Min,et al. Identification and Validation of Reference Genes for Quantitative Real-Time PCR Normalization and Its Applications in Lycium[J]. PLOS ONE,2014,9(5):13.
APA Zeng, Shaohua.,Liu, Yongliang.,Wu, Min.,Liu, Xiaomin.,Shen, Xiaofei.,...&Wang, Ying.(2014).Identification and Validation of Reference Genes for Quantitative Real-Time PCR Normalization and Its Applications in Lycium.PLOS ONE,9(5),13.
MLA Zeng, Shaohua,et al."Identification and Validation of Reference Genes for Quantitative Real-Time PCR Normalization and Its Applications in Lycium".PLOS ONE 9.5(2014):13.
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