Selection and Validation of Appropriate Reference Genes for Quantitative RT-PCR Analysis in Rubia yunnanensis Diels Based on Transcriptome Data
Real-time quantitative polymerase chain reaction (RT-qPCR) has been widely applied in gene expression and transcription abundance analysis because of its high sensitivity, good repeatability, and strong specificity. Selection of relatively stable reference genes is a precondition in order to obtain...
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| description | Real-time quantitative polymerase chain reaction (RT-qPCR) has been widely applied in gene expression and transcription abundance analysis because of its high sensitivity, good repeatability, and strong specificity. Selection of relatively stable reference genes is a precondition in order to obtain the reliable analysis results. However, little is known about evaluation of a set of reference genes through scientific experiments in Rubia plants. Here, 15 candidate reference genes were selected from R. yunnanensis transcriptome database and analyzed under abiotic stresses, hormone treatments, and different tissues. Among these 15 candidate reference genes, heterogeneous nuclear ribonucleoprotein (hnRNP), TATA binding protein (TBP), ribosomal protein L5 (RPL5), malate dehydrogenase (MDH), and elongation factor 1-alpha (EF-1α) were indicated as the five most stable reference genes by four statistical programs (geNorm, NormFinder, BestKeeper, and RefFinder). Ultimately, the validity of reference genes was confirmed by normalizing the expression of o-succinylbenzoate-CoA ligase (OSBL) and isochorismate synthase (ICS) involved in the anthraquinone biosynthesis pathway in different tissues and hormone treatments. Meanwhile, four other putative genes involved in the anthraquinone biosynthesis pathway were also normalized with the selected reference genes, which showed similar expression levels with those given by transcriptome data. This work is the first research that aims at a systematic validation on the stability of reference genes selected from R. yunnanensis transcriptome data and will be conducive to analyze gene expression in R. yunnanensis or other Rubia species. |
| doi_str_mv | 10.1155/2020/5824841 |
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Selection of relatively stable reference genes is a precondition in order to obtain the reliable analysis results. However, little is known about evaluation of a set of reference genes through scientific experiments in Rubia plants. Here, 15 candidate reference genes were selected from R. yunnanensis transcriptome database and analyzed under abiotic stresses, hormone treatments, and different tissues. Among these 15 candidate reference genes, heterogeneous nuclear ribonucleoprotein (hnRNP), TATA binding protein (TBP), ribosomal protein L5 (RPL5), malate dehydrogenase (MDH), and elongation factor 1-alpha (EF-1α) were indicated as the five most stable reference genes by four statistical programs (geNorm, NormFinder, BestKeeper, and RefFinder). Ultimately, the validity of reference genes was confirmed by normalizing the expression of o-succinylbenzoate-CoA ligase (OSBL) and isochorismate synthase (ICS) involved in the anthraquinone biosynthesis pathway in different tissues and hormone treatments. Meanwhile, four other putative genes involved in the anthraquinone biosynthesis pathway were also normalized with the selected reference genes, which showed similar expression levels with those given by transcriptome data. This work is the first research that aims at a systematic validation on the stability of reference genes selected from R. yunnanensis transcriptome data and will be conducive to analyze gene expression in R. yunnanensis or other Rubia species.</description><identifier>ISSN: 2314-6133</identifier><identifier>EISSN: 2314-6141</identifier><identifier>DOI: 10.1155/2020/5824841</identifier><identifier>PMID: 31998793</identifier><language>eng</language><publisher>Cairo, Egypt: Hindawi Publishing Corporation</publisher><subject>Anthraquinone ; Anthraquinones ; Biosynthesis ; Dehydrogenases ; Elongation ; Enzymes ; Gene expression ; Genes ; Genetic aspects ; Genetic transcription ; Isochorismate synthase ; Malate dehydrogenase ; Metabolism ; Normalizing ; O-Succinylbenzoate-CoA ligase ; Oxidative stress ; Plant sciences ; Polymerase chain reaction ; Protein binding ; Proteins ; Seeds ; Sensitivity analysis ; TATA-binding protein ; Transcription</subject><ispartof>BioMed research international, 2020, Vol.2020 (2020), p.1-19</ispartof><rights>Copyright © 2020 Shanyong Yi et al.</rights><rights>COPYRIGHT 2020 John Wiley & Sons, Inc.</rights><rights>Copyright © 2020 Shanyong Yi et al. This is an open access article distributed under the Creative Commons Attribution License (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License. http://creativecommons.org/licenses/by/4.0</rights><rights>Copyright © 2020 Shanyong Yi et al. 2020</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c499t-50935ab65dbe1a959d8e20d7da3f587379b22f0631f18739eeaf9879967fc9203</citedby><cites>FETCH-LOGICAL-c499t-50935ab65dbe1a959d8e20d7da3f587379b22f0631f18739eeaf9879967fc9203</cites><orcidid>0000-0001-6653-1515 ; 0000-0002-1690-7841 ; 0000-0002-3731-1000</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6973195/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6973195/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,881,4010,27900,27901,27902,53766,53768</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/31998793$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Khareedu, Venkateswara R.</contributor><contributor>Venkateswara R Khareedu</contributor><creatorcontrib>Miao, Yuanyuan</creatorcontrib><creatorcontrib>Wang, Jing</creatorcontrib><creatorcontrib>Zhang, Xuejia</creatorcontrib><creatorcontrib>Lin, Qianwen</creatorcontrib><creatorcontrib>Yi, Shanyong</creatorcontrib><creatorcontrib>Tan, Ninghua</creatorcontrib><title>Selection and Validation of Appropriate Reference Genes for Quantitative RT-PCR Analysis in Rubia yunnanensis Diels Based on Transcriptome Data</title><title>BioMed research international</title><addtitle>Biomed Res Int</addtitle><description>Real-time quantitative polymerase chain reaction (RT-qPCR) has been widely applied in gene expression and transcription abundance analysis because of its high sensitivity, good repeatability, and strong specificity. Selection of relatively stable reference genes is a precondition in order to obtain the reliable analysis results. However, little is known about evaluation of a set of reference genes through scientific experiments in Rubia plants. Here, 15 candidate reference genes were selected from R. yunnanensis transcriptome database and analyzed under abiotic stresses, hormone treatments, and different tissues. Among these 15 candidate reference genes, heterogeneous nuclear ribonucleoprotein (hnRNP), TATA binding protein (TBP), ribosomal protein L5 (RPL5), malate dehydrogenase (MDH), and elongation factor 1-alpha (EF-1α) were indicated as the five most stable reference genes by four statistical programs (geNorm, NormFinder, BestKeeper, and RefFinder). Ultimately, the validity of reference genes was confirmed by normalizing the expression of o-succinylbenzoate-CoA ligase (OSBL) and isochorismate synthase (ICS) involved in the anthraquinone biosynthesis pathway in different tissues and hormone treatments. Meanwhile, four other putative genes involved in the anthraquinone biosynthesis pathway were also normalized with the selected reference genes, which showed similar expression levels with those given by transcriptome data. This work is the first research that aims at a systematic validation on the stability of reference genes selected from R. yunnanensis transcriptome data and will be conducive to analyze gene expression in R. yunnanensis or other Rubia species.</description><subject>Anthraquinone</subject><subject>Anthraquinones</subject><subject>Biosynthesis</subject><subject>Dehydrogenases</subject><subject>Elongation</subject><subject>Enzymes</subject><subject>Gene expression</subject><subject>Genes</subject><subject>Genetic aspects</subject><subject>Genetic transcription</subject><subject>Isochorismate synthase</subject><subject>Malate dehydrogenase</subject><subject>Metabolism</subject><subject>Normalizing</subject><subject>O-Succinylbenzoate-CoA ligase</subject><subject>Oxidative stress</subject><subject>Plant sciences</subject><subject>Polymerase chain reaction</subject><subject>Protein binding</subject><subject>Proteins</subject><subject>Seeds</subject><subject>Sensitivity 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and Validation of Appropriate Reference Genes for Quantitative RT-PCR Analysis in Rubia yunnanensis Diels Based on Transcriptome Data</title><author>Miao, Yuanyuan ; Wang, Jing ; Zhang, Xuejia ; Lin, Qianwen ; Yi, Shanyong ; Tan, Ninghua</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c499t-50935ab65dbe1a959d8e20d7da3f587379b22f0631f18739eeaf9879967fc9203</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2020</creationdate><topic>Anthraquinone</topic><topic>Anthraquinones</topic><topic>Biosynthesis</topic><topic>Dehydrogenases</topic><topic>Elongation</topic><topic>Enzymes</topic><topic>Gene expression</topic><topic>Genes</topic><topic>Genetic aspects</topic><topic>Genetic transcription</topic><topic>Isochorismate synthase</topic><topic>Malate dehydrogenase</topic><topic>Metabolism</topic><topic>Normalizing</topic><topic>O-Succinylbenzoate-CoA ligase</topic><topic>Oxidative 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>BioMed research international</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Miao, Yuanyuan</au><au>Wang, Jing</au><au>Zhang, Xuejia</au><au>Lin, Qianwen</au><au>Yi, Shanyong</au><au>Tan, Ninghua</au><au>Khareedu, Venkateswara R.</au><au>Venkateswara R Khareedu</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Selection and Validation of Appropriate Reference Genes for Quantitative RT-PCR Analysis in Rubia yunnanensis Diels Based on Transcriptome Data</atitle><jtitle>BioMed research international</jtitle><addtitle>Biomed Res Int</addtitle><date>2020</date><risdate>2020</risdate><volume>2020</volume><issue>2020</issue><spage>1</spage><epage>19</epage><pages>1-19</pages><issn>2314-6133</issn><eissn>2314-6141</eissn><abstract>Real-time quantitative polymerase chain reaction (RT-qPCR) has been widely applied in gene expression and transcription abundance analysis because of its high sensitivity, good repeatability, and strong specificity. Selection of relatively stable reference genes is a precondition in order to obtain the reliable analysis results. However, little is known about evaluation of a set of reference genes through scientific experiments in Rubia plants. Here, 15 candidate reference genes were selected from R. yunnanensis transcriptome database and analyzed under abiotic stresses, hormone treatments, and different tissues. Among these 15 candidate reference genes, heterogeneous nuclear ribonucleoprotein (hnRNP), TATA binding protein (TBP), ribosomal protein L5 (RPL5), malate dehydrogenase (MDH), and elongation factor 1-alpha (EF-1α) were indicated as the five most stable reference genes by four statistical programs (geNorm, NormFinder, BestKeeper, and RefFinder). Ultimately, the validity of reference genes was confirmed by normalizing the expression of o-succinylbenzoate-CoA ligase (OSBL) and isochorismate synthase (ICS) involved in the anthraquinone biosynthesis pathway in different tissues and hormone treatments. Meanwhile, four other putative genes involved in the anthraquinone biosynthesis pathway were also normalized with the selected reference genes, which showed similar expression levels with those given by transcriptome data. This work is the first research that aims at a systematic validation on the stability of reference genes selected from R. yunnanensis transcriptome data and will be conducive to analyze gene expression in R. yunnanensis or other Rubia species.</abstract><cop>Cairo, Egypt</cop><pub>Hindawi Publishing Corporation</pub><pmid>31998793</pmid><doi>10.1155/2020/5824841</doi><tpages>19</tpages><orcidid>https://orcid.org/0000-0001-6653-1515</orcidid><orcidid>https://orcid.org/0000-0002-1690-7841</orcidid><orcidid>https://orcid.org/0000-0002-3731-1000</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | Anthraquinone Anthraquinones Biosynthesis Dehydrogenases Elongation Enzymes Gene expression Genes Genetic aspects Genetic transcription Isochorismate synthase Malate dehydrogenase Metabolism Normalizing O-Succinylbenzoate-CoA ligase Oxidative stress Plant sciences Polymerase chain reaction Protein binding Proteins Seeds Sensitivity analysis TATA-binding protein Transcription |
| title | Selection and Validation of Appropriate Reference Genes for Quantitative RT-PCR Analysis in Rubia yunnanensis Diels Based on Transcriptome Data |
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