Selection and validation of reference genes for quantitative gene expression analyses in various tissues and seeds at different developmental stages in Bixa orellana L
Bixa orellana L., popularly known as annatto, produces several secondary metabolites of pharmaceutical and industrial interest, including bixin, whose molecular basis of biosynthesis remain to be determined. Gene expression analysis by quantitative real-time PCR (qPCR) is an important tool to advanc...
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| Veröffentlicht in: | Physiology and molecular biology of plants 2018-05, Vol.24 (3), p.369-378 |
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| container_title | Physiology and molecular biology of plants |
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| creator | Moreira, Viviane S. Soares, Virgínia L. F. Silva, Raner J. S. Sousa, Aurizangela O. Otoni, Wagner C. Costa, Marcio G. C. |
| description | Bixa orellana
L., popularly known as annatto, produces several secondary metabolites of pharmaceutical and industrial interest, including bixin, whose molecular basis of biosynthesis remain to be determined. Gene expression analysis by quantitative real-time PCR (qPCR) is an important tool to advance such knowledge. However, correct interpretation of qPCR data requires the use of suitable reference genes in order to reduce experimental variations. In the present study, we have selected four different candidates for reference genes in
B. orellana
, coding for 40S ribosomal protein S9 (RPS9), histone H4 (H4), 60S ribosomal protein L38 (RPL38) and 18S ribosomal RNA (18SrRNA). Their expression stabilities in different tissues (e.g. flower buds, flowers, leaves and seeds at different developmental stages) were analyzed using five statistical tools (NormFinder, geNorm, BestKeeper, ΔCt method and RefFinder). The results indicated that
RPL38
is the most stable gene in different tissues and stages of seed development and
18SrRNA
is the most unstable among the analyzed genes. In order to validate the candidate reference genes, we have analyzed the relative expression of a target gene coding for carotenoid cleavage dioxygenase 1 (CCD1) using the stable
RPL38
and the least stable gene,
18SrRNA
, for normalization of the qPCR data. The results demonstrated significant differences in the interpretation of the
CCD1
gene expression data, depending on the reference gene used, reinforcing the importance of the correct selection of reference genes for normalization. |
| doi_str_mv | 10.1007/s12298-018-0528-1 |
| format | Article |
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L., popularly known as annatto, produces several secondary metabolites of pharmaceutical and industrial interest, including bixin, whose molecular basis of biosynthesis remain to be determined. Gene expression analysis by quantitative real-time PCR (qPCR) is an important tool to advance such knowledge. However, correct interpretation of qPCR data requires the use of suitable reference genes in order to reduce experimental variations. In the present study, we have selected four different candidates for reference genes in
B. orellana
, coding for 40S ribosomal protein S9 (RPS9), histone H4 (H4), 60S ribosomal protein L38 (RPL38) and 18S ribosomal RNA (18SrRNA). Their expression stabilities in different tissues (e.g. flower buds, flowers, leaves and seeds at different developmental stages) were analyzed using five statistical tools (NormFinder, geNorm, BestKeeper, ΔCt method and RefFinder). The results indicated that
RPL38
is the most stable gene in different tissues and stages of seed development and
18SrRNA
is the most unstable among the analyzed genes. In order to validate the candidate reference genes, we have analyzed the relative expression of a target gene coding for carotenoid cleavage dioxygenase 1 (CCD1) using the stable
RPL38
and the least stable gene,
18SrRNA
, for normalization of the qPCR data. The results demonstrated significant differences in the interpretation of the
CCD1
gene expression data, depending on the reference gene used, reinforcing the importance of the correct selection of reference genes for normalization.</description><identifier>ISSN: 0971-5894</identifier><identifier>EISSN: 0974-0430</identifier><identifier>DOI: 10.1007/s12298-018-0528-1</identifier><identifier>PMID: 29692545</identifier><language>eng</language><publisher>New Delhi: Springer India</publisher><subject>Annatto ; Biological and Medical Physics ; Biomedical and Life Sciences ; Biophysics ; Biosynthesis ; Bixa orellana ; buds ; carotenoids ; Cell Biology ; Developmental stages ; Dioxygenase ; Flowers ; Gene expression ; Genes ; Histone H4 ; histones ; leaves ; Life Sciences ; Metabolites ; Plant Physiology ; Plant Sciences ; Plant tissues ; quantitative polymerase chain reaction ; Research Article ; Ribonucleic acid ; Ribosomal protein S9 ; ribosomal proteins ; ribosomal RNA ; RNA ; rRNA 18S ; Secondary metabolites ; seed development ; Seeds ; tissues</subject><ispartof>Physiology and molecular biology of plants, 2018-05, Vol.24 (3), p.369-378</ispartof><rights>Prof. H.S. Srivastava Foundation for Science and Society 2018</rights><rights>Copyright Springer Science & Business Media 2018</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c503t-10926207c5600cec6f3767699a72f693cd7c4613d03c377f590418bd9479eafd3</citedby><cites>FETCH-LOGICAL-c503t-10926207c5600cec6f3767699a72f693cd7c4613d03c377f590418bd9479eafd3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5911269/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5911269/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,723,776,780,881,27901,27902,41464,42533,51294,53766,53768</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/29692545$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Moreira, Viviane S.</creatorcontrib><creatorcontrib>Soares, Virgínia L. F.</creatorcontrib><creatorcontrib>Silva, Raner J. S.</creatorcontrib><creatorcontrib>Sousa, Aurizangela O.</creatorcontrib><creatorcontrib>Otoni, Wagner C.</creatorcontrib><creatorcontrib>Costa, Marcio G. C.</creatorcontrib><title>Selection and validation of reference genes for quantitative gene expression analyses in various tissues and seeds at different developmental stages in Bixa orellana L</title><title>Physiology and molecular biology of plants</title><addtitle>Physiol Mol Biol Plants</addtitle><addtitle>Physiol Mol Biol Plants</addtitle><description>Bixa orellana
L., popularly known as annatto, produces several secondary metabolites of pharmaceutical and industrial interest, including bixin, whose molecular basis of biosynthesis remain to be determined. Gene expression analysis by quantitative real-time PCR (qPCR) is an important tool to advance such knowledge. However, correct interpretation of qPCR data requires the use of suitable reference genes in order to reduce experimental variations. In the present study, we have selected four different candidates for reference genes in
B. orellana
, coding for 40S ribosomal protein S9 (RPS9), histone H4 (H4), 60S ribosomal protein L38 (RPL38) and 18S ribosomal RNA (18SrRNA). Their expression stabilities in different tissues (e.g. flower buds, flowers, leaves and seeds at different developmental stages) were analyzed using five statistical tools (NormFinder, geNorm, BestKeeper, ΔCt method and RefFinder). The results indicated that
RPL38
is the most stable gene in different tissues and stages of seed development and
18SrRNA
is the most unstable among the analyzed genes. In order to validate the candidate reference genes, we have analyzed the relative expression of a target gene coding for carotenoid cleavage dioxygenase 1 (CCD1) using the stable
RPL38
and the least stable gene,
18SrRNA
, for normalization of the qPCR data. The results demonstrated significant differences in the interpretation of the
CCD1
gene expression data, depending on the reference gene used, reinforcing the importance of the correct selection of reference genes for normalization.</description><subject>Annatto</subject><subject>Biological and Medical Physics</subject><subject>Biomedical and Life Sciences</subject><subject>Biophysics</subject><subject>Biosynthesis</subject><subject>Bixa orellana</subject><subject>buds</subject><subject>carotenoids</subject><subject>Cell Biology</subject><subject>Developmental stages</subject><subject>Dioxygenase</subject><subject>Flowers</subject><subject>Gene expression</subject><subject>Genes</subject><subject>Histone H4</subject><subject>histones</subject><subject>leaves</subject><subject>Life Sciences</subject><subject>Metabolites</subject><subject>Plant Physiology</subject><subject>Plant Sciences</subject><subject>Plant tissues</subject><subject>quantitative polymerase chain reaction</subject><subject>Research Article</subject><subject>Ribonucleic acid</subject><subject>Ribosomal protein S9</subject><subject>ribosomal proteins</subject><subject>ribosomal RNA</subject><subject>RNA</subject><subject>rRNA 18S</subject><subject>Secondary metabolites</subject><subject>seed development</subject><subject>Seeds</subject><subject>tissues</subject><issn>0971-5894</issn><issn>0974-0430</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2018</creationdate><recordtype>article</recordtype><recordid>eNqNkk1v1DAQhiMEomXhB3BBlrhwCYw_YscXJFrxJa3EAThbrjNZXGXjrZ2s2l_E32R2U8qHhMTB8th-5h2P_VbVUw4vOYB5VbgQtq2B02hEW_N71SlYo2pQEu4fY143rVUn1aNSLgG0VIY_rE6E1VY0qjmtvn_GAcMU08j82LG9H2Lnj8vUs4w9ZhwDsg2OWFifMrua_TjFiZj9ss3wepexlEXCDzeFyDiSVI5pLmyKpcy0dZAviB1FE-tif5SmCPc4pN2WYj-wMvnNkn4Wrz1LGYeBRNn6cfWg90PBJ7fzqvr67u2X8w_1-tP7j-dv1nVoQE41Byu0ABMaDRAw6F4abbS13oheWxk6E5TmsgMZpDF9Y0Hx9qKzylj0fSdX1etFdzdfbLELdK3sB7fLcevzjUs-uj9PxvjNbdLeNZZzQRVW1YtbgZyuqO_JbWMJxzaQnsMJ0Ea0oDT8ByrBSiPBEPr8L_QyzZle-0CJVrWa-iWKL1TIqRT6vbt7c3AHx7jFMY4c4w6OcZxynv3e8F3GT4sQIBag0NG4wfyr9L9VfwA_N88P</recordid><startdate>20180501</startdate><enddate>20180501</enddate><creator>Moreira, Viviane S.</creator><creator>Soares, Virgínia L. F.</creator><creator>Silva, Raner J. S.</creator><creator>Sousa, Aurizangela O.</creator><creator>Otoni, Wagner C.</creator><creator>Costa, Marcio G. C.</creator><general>Springer India</general><general>Springer Nature B.V</general><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>7S9</scope><scope>L.6</scope><scope>5PM</scope></search><sort><creationdate>20180501</creationdate><title>Selection and validation of reference genes for quantitative gene expression analyses in various tissues and seeds at different developmental stages in Bixa orellana L</title><author>Moreira, Viviane S. ; Soares, Virgínia L. F. ; Silva, Raner J. S. ; Sousa, Aurizangela O. ; Otoni, Wagner C. ; Costa, Marcio G. C.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c503t-10926207c5600cec6f3767699a72f693cd7c4613d03c377f590418bd9479eafd3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2018</creationdate><topic>Annatto</topic><topic>Biological and Medical Physics</topic><topic>Biomedical and Life Sciences</topic><topic>Biophysics</topic><topic>Biosynthesis</topic><topic>Bixa orellana</topic><topic>buds</topic><topic>carotenoids</topic><topic>Cell Biology</topic><topic>Developmental stages</topic><topic>Dioxygenase</topic><topic>Flowers</topic><topic>Gene expression</topic><topic>Genes</topic><topic>Histone H4</topic><topic>histones</topic><topic>leaves</topic><topic>Life Sciences</topic><topic>Metabolites</topic><topic>Plant Physiology</topic><topic>Plant Sciences</topic><topic>Plant tissues</topic><topic>quantitative polymerase chain reaction</topic><topic>Research Article</topic><topic>Ribonucleic acid</topic><topic>Ribosomal protein S9</topic><topic>ribosomal proteins</topic><topic>ribosomal RNA</topic><topic>RNA</topic><topic>rRNA 18S</topic><topic>Secondary metabolites</topic><topic>seed development</topic><topic>Seeds</topic><topic>tissues</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Moreira, Viviane S.</creatorcontrib><creatorcontrib>Soares, Virgínia L. F.</creatorcontrib><creatorcontrib>Silva, Raner J. S.</creatorcontrib><creatorcontrib>Sousa, Aurizangela O.</creatorcontrib><creatorcontrib>Otoni, Wagner C.</creatorcontrib><creatorcontrib>Costa, Marcio G. C.</creatorcontrib><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>AGRICOLA</collection><collection>AGRICOLA - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Physiology and molecular biology of plants</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Moreira, Viviane S.</au><au>Soares, Virgínia L. F.</au><au>Silva, Raner J. S.</au><au>Sousa, Aurizangela O.</au><au>Otoni, Wagner C.</au><au>Costa, Marcio G. C.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Selection and validation of reference genes for quantitative gene expression analyses in various tissues and seeds at different developmental stages in Bixa orellana L</atitle><jtitle>Physiology and molecular biology of plants</jtitle><stitle>Physiol Mol Biol Plants</stitle><addtitle>Physiol Mol Biol Plants</addtitle><date>2018-05-01</date><risdate>2018</risdate><volume>24</volume><issue>3</issue><spage>369</spage><epage>378</epage><pages>369-378</pages><issn>0971-5894</issn><eissn>0974-0430</eissn><abstract>Bixa orellana
L., popularly known as annatto, produces several secondary metabolites of pharmaceutical and industrial interest, including bixin, whose molecular basis of biosynthesis remain to be determined. Gene expression analysis by quantitative real-time PCR (qPCR) is an important tool to advance such knowledge. However, correct interpretation of qPCR data requires the use of suitable reference genes in order to reduce experimental variations. In the present study, we have selected four different candidates for reference genes in
B. orellana
, coding for 40S ribosomal protein S9 (RPS9), histone H4 (H4), 60S ribosomal protein L38 (RPL38) and 18S ribosomal RNA (18SrRNA). Their expression stabilities in different tissues (e.g. flower buds, flowers, leaves and seeds at different developmental stages) were analyzed using five statistical tools (NormFinder, geNorm, BestKeeper, ΔCt method and RefFinder). The results indicated that
RPL38
is the most stable gene in different tissues and stages of seed development and
18SrRNA
is the most unstable among the analyzed genes. In order to validate the candidate reference genes, we have analyzed the relative expression of a target gene coding for carotenoid cleavage dioxygenase 1 (CCD1) using the stable
RPL38
and the least stable gene,
18SrRNA
, for normalization of the qPCR data. The results demonstrated significant differences in the interpretation of the
CCD1
gene expression data, depending on the reference gene used, reinforcing the importance of the correct selection of reference genes for normalization.</abstract><cop>New Delhi</cop><pub>Springer India</pub><pmid>29692545</pmid><doi>10.1007/s12298-018-0528-1</doi><tpages>10</tpages><oa>free_for_read</oa></addata></record> |
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| source | Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central; SpringerLink Journals - AutoHoldings |
| subjects | Annatto Biological and Medical Physics Biomedical and Life Sciences Biophysics Biosynthesis Bixa orellana buds carotenoids Cell Biology Developmental stages Dioxygenase Flowers Gene expression Genes Histone H4 histones leaves Life Sciences Metabolites Plant Physiology Plant Sciences Plant tissues quantitative polymerase chain reaction Research Article Ribonucleic acid Ribosomal protein S9 ribosomal proteins ribosomal RNA RNA rRNA 18S Secondary metabolites seed development Seeds tissues |
| title | Selection and validation of reference genes for quantitative gene expression analyses in various tissues and seeds at different developmental stages in Bixa orellana L |
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