Validation of suitable reference genes for expression normalization in Echinococcus spp. larval stages
In recent years, a significant amount of sequence data (both genomic and transcriptomic) for Echinococcus spp. has been published, thereby facilitating the analysis of genes expressed during a specific stage or involved in parasite development. To perform a suitable gene expression quantification an...
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| description | In recent years, a significant amount of sequence data (both genomic and transcriptomic) for Echinococcus spp. has been published, thereby facilitating the analysis of genes expressed during a specific stage or involved in parasite development. To perform a suitable gene expression quantification analysis, the use of validated reference genes is strongly recommended. Thus, the aim of this work was to identify suitable reference genes to allow reliable expression normalization for genes of interest in Echinococcus granulosus sensu stricto (s.s.) (G1) and Echinococcus ortleppi upon induction of the early pre-adult development. Untreated protoscoleces (PS) and pepsin-treated protoscoleces (PSP) from E. granulosus s.s. (G1) and E. ortleppi metacestode were used. The gene expression stability of eleven candidate reference genes (βTUB, NDUFV2, RPL13, TBP, CYP-1, RPII, EF-1α, βACT-1, GAPDH, ETIF4A-III and MAPK3) was assessed using geNorm, Normfinder, and RefFinder. Our qPCR data showed a good correlation with the recently published RNA-seq data. Regarding expression stability, EF-1α and TBP were the most stable genes for both species. Interestingly, βACT-1 (the most commonly used reference gene), and GAPDH and ETIF4A-III (previously identified as housekeeping genes) did not behave stably in our assay conditions. We propose the use of EF-1α as a reference gene for studies involving gene expression analysis in both PS and PSP experimental conditions for E. granulosus s.s. and E. ortleppi. To demonstrate its applicability, EF-1α was used as a normalizer gene in the relative quantification of transcripts from genes coding for antigen B subunits. The same EF-1α reference gene may be used in studies with other Echinococcus sensu lato species. This report validates suitable reference genes for species of class Cestoda, phylum Platyhelminthes, thus providing a foundation for further validation in other epidemiologically important cestode species, such as those from the Taenia genus. |
| doi_str_mv | 10.1371/journal.pone.0102228 |
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To perform a suitable gene expression quantification analysis, the use of validated reference genes is strongly recommended. Thus, the aim of this work was to identify suitable reference genes to allow reliable expression normalization for genes of interest in Echinococcus granulosus sensu stricto (s.s.) (G1) and Echinococcus ortleppi upon induction of the early pre-adult development. Untreated protoscoleces (PS) and pepsin-treated protoscoleces (PSP) from E. granulosus s.s. (G1) and E. ortleppi metacestode were used. The gene expression stability of eleven candidate reference genes (βTUB, NDUFV2, RPL13, TBP, CYP-1, RPII, EF-1α, βACT-1, GAPDH, ETIF4A-III and MAPK3) was assessed using geNorm, Normfinder, and RefFinder. Our qPCR data showed a good correlation with the recently published RNA-seq data. Regarding expression stability, EF-1α and TBP were the most stable genes for both species. Interestingly, βACT-1 (the most commonly used reference gene), and GAPDH and ETIF4A-III (previously identified as housekeeping genes) did not behave stably in our assay conditions. We propose the use of EF-1α as a reference gene for studies involving gene expression analysis in both PS and PSP experimental conditions for E. granulosus s.s. and E. ortleppi. To demonstrate its applicability, EF-1α was used as a normalizer gene in the relative quantification of transcripts from genes coding for antigen B subunits. The same EF-1α reference gene may be used in studies with other Echinococcus sensu lato species. This report validates suitable reference genes for species of class Cestoda, phylum Platyhelminthes, thus providing a foundation for further validation in other epidemiologically important cestode species, such as those from the Taenia genus.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0102228</identifier><identifier>PMID: 25014071</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Analysis ; Animals ; Antigens ; Biology and Life Sciences ; Cattle ; Cysts ; Echinococcosis - parasitology ; Echinococcus ; Echinococcus - genetics ; Echinococcus - growth & development ; Echinococcus - isolation & purification ; Echinococcus granulosus ; Echinococcus granulosus - genetics ; Echinococcus granulosus - growth & development ; Echinococcus granulosus - isolation & purification ; Echinococcus multilocularis ; Epidemiology ; Gene expression ; Gene Expression Profiling ; Gene Expression Regulation, Developmental ; Genes ; Genes, Essential ; Genes, Helminth ; Genomes ; Glyceraldehyde-3-phosphate dehydrogenase ; Life Cycle Stages - genetics ; Lipoproteins - genetics ; Parasites ; Pepsin ; Peptide Elongation Factor 1 - genetics ; Proteomics ; Public health ; Ribonucleic acid ; RNA ; RNA, Helminth - genetics ; RNA, Messenger - genetics ; Species ; Stability</subject><ispartof>PloS one, 2014-07, Vol.9 (7), p.e102228-e102228</ispartof><rights>COPYRIGHT 2014 Public Library of Science</rights><rights>2014 Espínola et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2014 Espínola et al 2014 Espínola et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c692t-1f39d2f592bb94e73d65a1e4d0e17d2b08fd9f24ba1b73d3e107826bc43fc0b23</citedby><cites>FETCH-LOGICAL-c692t-1f39d2f592bb94e73d65a1e4d0e17d2b08fd9f24ba1b73d3e107826bc43fc0b23</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/PMC4094502/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4094502/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,2102,2928,23866,27924,27925,53791,53793,79600,79601</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/25014071$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Schönbach, Christian</contributor><creatorcontrib>Espínola, Sergio Martin</creatorcontrib><creatorcontrib>Ferreira, Henrique Bunselmeyer</creatorcontrib><creatorcontrib>Zaha, Arnaldo</creatorcontrib><title>Validation of suitable reference genes for expression normalization in Echinococcus spp. larval stages</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>In recent years, a significant amount of sequence data (both genomic and transcriptomic) for Echinococcus spp. has been published, thereby facilitating the analysis of genes expressed during a specific stage or involved in parasite development. 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Interestingly, βACT-1 (the most commonly used reference gene), and GAPDH and ETIF4A-III (previously identified as housekeeping genes) did not behave stably in our assay conditions. We propose the use of EF-1α as a reference gene for studies involving gene expression analysis in both PS and PSP experimental conditions for E. granulosus s.s. and E. ortleppi. To demonstrate its applicability, EF-1α was used as a normalizer gene in the relative quantification of transcripts from genes coding for antigen B subunits. The same EF-1α reference gene may be used in studies with other Echinococcus sensu lato species. 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genetics</subject><subject>Lipoproteins - genetics</subject><subject>Parasites</subject><subject>Pepsin</subject><subject>Peptide Elongation Factor 1 - genetics</subject><subject>Proteomics</subject><subject>Public health</subject><subject>Ribonucleic acid</subject><subject>RNA</subject><subject>RNA, Helminth - genetics</subject><subject>RNA, Messenger - genetics</subject><subject>Species</subject><subject>Stability</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2014</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><sourceid>DOA</sourceid><recordid>eNqNk11rFDEUhgdRbK3-A9EBQfRix3zN141QStWFQsGP3oZM5mQ2SzaZJjOl-uvNdKdlR3ohuUhInvc9yck5SfIaowzTEn_autFbYbLeWcgQRoSQ6klyjGtKVgVB9OnB-ih5EcIWoZxWRfE8OSI5wgyV-DhRV8LoVgza2dSpNIx6EI2B1IMCD1ZC2oGFkCrnU7jtPYQwodb5XRT-2Qu1Tc_lRlsnnZRjSEPfZ6kR_kaYNAyig_AyeaaECfBqnk-SX1_Of559W11cfl2fnV6sZFGTYYUVrVui8po0Tc2gpG2RCwysRYDLljSoUm2tCGsEbuIhBYzKihSNZFRJ1BB6krzd-_bGBT6nKHCcM5ZjimkdifWeaJ3Y8t7rnfC_uROa320433HhBy0NcEGjhmFGJCOsLMuG4JrULG-QLKBUk9fnOdrY7KCVYAcvzMJ0eWL1hnfuhjMUbdB03Q-zgXfXI4SB73SQYIyw4Ma7e-e4QkU9xXr3D_r462aqE_EB2ioX48rJlJ8yXBU0j1CkskeoOFrYaRnrSem4vxB8XAgiM8Dt0IkxBL7-8f3_2curJfv-gN2AMMMmODNOVRWWINuD0rsQYnE-JBkjPrXDfTb41A58bocoe3P4QQ-i-_qnfwGwsQUY</recordid><startdate>20140711</startdate><enddate>20140711</enddate><creator>Espínola, Sergio Martin</creator><creator>Ferreira, Henrique Bunselmeyer</creator><creator>Zaha, Arnaldo</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>IOV</scope><scope>ISR</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QO</scope><scope>7RV</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TG</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>KB0</scope><scope>KL.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>NAPCQ</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PATMY</scope><scope>PDBOC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope></search><sort><creationdate>20140711</creationdate><title>Validation of suitable reference genes for expression normalization in Echinococcus spp. larval stages</title><author>Espínola, Sergio Martin ; Ferreira, Henrique Bunselmeyer ; Zaha, Arnaldo</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c692t-1f39d2f592bb94e73d65a1e4d0e17d2b08fd9f24ba1b73d3e107826bc43fc0b23</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2014</creationdate><topic>Analysis</topic><topic>Animals</topic><topic>Antigens</topic><topic>Biology and Life Sciences</topic><topic>Cattle</topic><topic>Cysts</topic><topic>Echinococcosis - parasitology</topic><topic>Echinococcus</topic><topic>Echinococcus - genetics</topic><topic>Echinococcus - growth & development</topic><topic>Echinococcus - isolation & purification</topic><topic>Echinococcus granulosus</topic><topic>Echinococcus granulosus - genetics</topic><topic>Echinococcus granulosus - growth & development</topic><topic>Echinococcus granulosus - isolation & purification</topic><topic>Echinococcus multilocularis</topic><topic>Epidemiology</topic><topic>Gene expression</topic><topic>Gene Expression Profiling</topic><topic>Gene Expression Regulation, Developmental</topic><topic>Genes</topic><topic>Genes, Essential</topic><topic>Genes, Helminth</topic><topic>Genomes</topic><topic>Glyceraldehyde-3-phosphate dehydrogenase</topic><topic>Life Cycle Stages - genetics</topic><topic>Lipoproteins - genetics</topic><topic>Parasites</topic><topic>Pepsin</topic><topic>Peptide Elongation Factor 1 - genetics</topic><topic>Proteomics</topic><topic>Public health</topic><topic>Ribonucleic acid</topic><topic>RNA</topic><topic>RNA, Helminth - 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Espínola, Sergio Martin</au><au>Ferreira, Henrique Bunselmeyer</au><au>Zaha, Arnaldo</au><au>Schönbach, Christian</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Validation of suitable reference genes for expression normalization in Echinococcus spp. larval stages</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2014-07-11</date><risdate>2014</risdate><volume>9</volume><issue>7</issue><spage>e102228</spage><epage>e102228</epage><pages>e102228-e102228</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>In recent years, a significant amount of sequence data (both genomic and transcriptomic) for Echinococcus spp. has been published, thereby facilitating the analysis of genes expressed during a specific stage or involved in parasite development. To perform a suitable gene expression quantification analysis, the use of validated reference genes is strongly recommended. Thus, the aim of this work was to identify suitable reference genes to allow reliable expression normalization for genes of interest in Echinococcus granulosus sensu stricto (s.s.) (G1) and Echinococcus ortleppi upon induction of the early pre-adult development. Untreated protoscoleces (PS) and pepsin-treated protoscoleces (PSP) from E. granulosus s.s. (G1) and E. ortleppi metacestode were used. The gene expression stability of eleven candidate reference genes (βTUB, NDUFV2, RPL13, TBP, CYP-1, RPII, EF-1α, βACT-1, GAPDH, ETIF4A-III and MAPK3) was assessed using geNorm, Normfinder, and RefFinder. Our qPCR data showed a good correlation with the recently published RNA-seq data. Regarding expression stability, EF-1α and TBP were the most stable genes for both species. Interestingly, βACT-1 (the most commonly used reference gene), and GAPDH and ETIF4A-III (previously identified as housekeeping genes) did not behave stably in our assay conditions. We propose the use of EF-1α as a reference gene for studies involving gene expression analysis in both PS and PSP experimental conditions for E. granulosus s.s. and E. ortleppi. To demonstrate its applicability, EF-1α was used as a normalizer gene in the relative quantification of transcripts from genes coding for antigen B subunits. The same EF-1α reference gene may be used in studies with other Echinococcus sensu lato species. This report validates suitable reference genes for species of class Cestoda, phylum Platyhelminthes, thus providing a foundation for further validation in other epidemiologically important cestode species, such as those from the Taenia genus.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>25014071</pmid><doi>10.1371/journal.pone.0102228</doi><oa>free_for_read</oa></addata></record> |
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| subjects | Analysis Animals Antigens Biology and Life Sciences Cattle Cysts Echinococcosis - parasitology Echinococcus Echinococcus - genetics Echinococcus - growth & development Echinococcus - isolation & purification Echinococcus granulosus Echinococcus granulosus - genetics Echinococcus granulosus - growth & development Echinococcus granulosus - isolation & purification Echinococcus multilocularis Epidemiology Gene expression Gene Expression Profiling Gene Expression Regulation, Developmental Genes Genes, Essential Genes, Helminth Genomes Glyceraldehyde-3-phosphate dehydrogenase Life Cycle Stages - genetics Lipoproteins - genetics Parasites Pepsin Peptide Elongation Factor 1 - genetics Proteomics Public health Ribonucleic acid RNA RNA, Helminth - genetics RNA, Messenger - genetics Species Stability |
| title | Validation of suitable reference genes for expression normalization in Echinococcus spp. larval stages |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2024-12-29T03%3A02%3A51IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Validation%20of%20suitable%20reference%20genes%20for%20expression%20normalization%20in%20Echinococcus%20spp.%20larval%20stages&rft.jtitle=PloS%20one&rft.au=Esp%C3%ADnola,%20Sergio%20Martin&rft.date=2014-07-11&rft.volume=9&rft.issue=7&rft.spage=e102228&rft.epage=e102228&rft.pages=e102228-e102228&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0102228&rft_dat=%3Cgale_plos_%3EA418635393%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=1544513139&rft_id=info:pmid/25014071&rft_galeid=A418635393&rft_doaj_id=oai_doaj_org_article_a35134142c424777b2192945b0c6e7f9&rfr_iscdi=true |