Evaluation of reference genes for gene expression analysis by real-time quantitative PCR (qPCR) in three stingless bee species (Hymenoptera: Apidae: Meliponini)
Stingless bees are generalist pollinators distributed through the pantropical region. There is growing evidence that their wild populations are experiencing substantial decline in response to habitat degradation and pesticides. Policies for conservation of endangered species will benefit from studie...
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creator | Freitas, Flávia C. P. Depintor, Thiago S. Agostini, Lucas T. Luna-Lucena, Danielle Nunes, Francis M. F. Bitondi, Márcia M. G. Simões, Zilá L. P. Lourenço, Anete P. |
description | Stingless bees are generalist pollinators distributed through the pantropical region. There is growing evidence that their wild populations are experiencing substantial decline in response to habitat degradation and pesticides. Policies for conservation of endangered species will benefit from studies focusing on genetic and molecular aspects of their development and behavior. The most common method for looking at gene expression is real-time quantitative polymerase chain reaction preceded by reverse transcription (RT-qPCR) of the mRNA of interest. This method requires the identification of reliable reference genes to correctly estimate fluctuations in transcript levels. To contribute to molecular studies on stingless bees, we used
Frieseomelitta varia
,
Melipona quadrifasciata
, and
Scaptotrigona bipunctata
species to test the expression stability of eight reference genes (
act
,
ef1-α
,
gapdh
,
rpl32
,
rps5
,
rps18
,
tbp
, and
tbp-af
) in RT-qPCR procedures in five physiological and experimental conditions (development, sex, tissues, bacteria injection, and pesticide exposure). In general, the
rpl32
,
rps5
and
rps18
ribosomal protein genes and
tpb-af
gene showed the highest stability, thus being identified as suitable reference genes for the three stingless bee species and defined conditions. Our results also emphasized the need to evaluate the stability of candidate genes for any designed experimental condition and stingless bee species. |
doi_str_mv | 10.1038/s41598-019-53544-0 |
format | Article |
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Frieseomelitta varia
,
Melipona quadrifasciata
, and
Scaptotrigona bipunctata
species to test the expression stability of eight reference genes (
act
,
ef1-α
,
gapdh
,
rpl32
,
rps5
,
rps18
,
tbp
, and
tbp-af
) in RT-qPCR procedures in five physiological and experimental conditions (development, sex, tissues, bacteria injection, and pesticide exposure). In general, the
rpl32
,
rps5
and
rps18
ribosomal protein genes and
tpb-af
gene showed the highest stability, thus being identified as suitable reference genes for the three stingless bee species and defined conditions. Our results also emphasized the need to evaluate the stability of candidate genes for any designed experimental condition and stingless bee species.</description><identifier>ISSN: 2045-2322</identifier><identifier>EISSN: 2045-2322</identifier><identifier>DOI: 10.1038/s41598-019-53544-0</identifier><identifier>PMID: 31776359</identifier><language>eng</language><publisher>London: Nature Publishing Group UK</publisher><subject>38/77 ; 631/1647/2017 ; 631/337/572 ; Animals ; Bees ; Bees - classification ; Bees - genetics ; Bees - growth & development ; Bees - microbiology ; Biodegradation ; Endangered species ; Environmental degradation ; Escherichia coli ; Escherichia coli Infections - genetics ; Escherichia coli Infections - microbiology ; Fat Body ; Female ; Gene expression ; Gene Expression - drug effects ; Genes, Essential ; Glyceraldehyde-3-phosphate dehydrogenase ; Head ; Humanities and Social Sciences ; Larva - genetics ; Male ; multidisciplinary ; Ovary ; Pesticides ; Pesticides - pharmacology ; Pollinators ; Polymerase chain reaction ; Pupa - genetics ; Real-Time Polymerase Chain Reaction - methods ; Reverse transcription ; Science ; Science (multidisciplinary) ; Sex</subject><ispartof>Scientific reports, 2019-11, Vol.9 (1), p.17692-13, Article 17692</ispartof><rights>The Author(s) 2019</rights><rights>2019. This work is published under http://creativecommons.org/licenses/by/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c540t-9085d37a5bdd71a3bb667c9e625e66ddffb3629d7dc99c84e4348e3f8288f1913</citedby><cites>FETCH-LOGICAL-c540t-9085d37a5bdd71a3bb667c9e625e66ddffb3629d7dc99c84e4348e3f8288f1913</cites><orcidid>0000-0001-6929-7277 ; 0000-0002-3162-4890</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/PMC6881334/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6881334/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,27901,27902,41096,42165,51551,53766,53768</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/31776359$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Freitas, Flávia C. P.</creatorcontrib><creatorcontrib>Depintor, Thiago S.</creatorcontrib><creatorcontrib>Agostini, Lucas T.</creatorcontrib><creatorcontrib>Luna-Lucena, Danielle</creatorcontrib><creatorcontrib>Nunes, Francis M. F.</creatorcontrib><creatorcontrib>Bitondi, Márcia M. G.</creatorcontrib><creatorcontrib>Simões, Zilá L. P.</creatorcontrib><creatorcontrib>Lourenço, Anete P.</creatorcontrib><title>Evaluation of reference genes for gene expression analysis by real-time quantitative PCR (qPCR) in three stingless bee species (Hymenoptera: Apidae: Meliponini)</title><title>Scientific reports</title><addtitle>Sci Rep</addtitle><addtitle>Sci Rep</addtitle><description>Stingless bees are generalist pollinators distributed through the pantropical region. There is growing evidence that their wild populations are experiencing substantial decline in response to habitat degradation and pesticides. Policies for conservation of endangered species will benefit from studies focusing on genetic and molecular aspects of their development and behavior. The most common method for looking at gene expression is real-time quantitative polymerase chain reaction preceded by reverse transcription (RT-qPCR) of the mRNA of interest. This method requires the identification of reliable reference genes to correctly estimate fluctuations in transcript levels. To contribute to molecular studies on stingless bees, we used
Frieseomelitta varia
,
Melipona quadrifasciata
, and
Scaptotrigona bipunctata
species to test the expression stability of eight reference genes (
act
,
ef1-α
,
gapdh
,
rpl32
,
rps5
,
rps18
,
tbp
, and
tbp-af
) in RT-qPCR procedures in five physiological and experimental conditions (development, sex, tissues, bacteria injection, and pesticide exposure). In general, the
rpl32
,
rps5
and
rps18
ribosomal protein genes and
tpb-af
gene showed the highest stability, thus being identified as suitable reference genes for the three stingless bee species and defined conditions. Our results also emphasized the need to evaluate the stability of candidate genes for any designed experimental condition and stingless bee species.</description><subject>38/77</subject><subject>631/1647/2017</subject><subject>631/337/572</subject><subject>Animals</subject><subject>Bees</subject><subject>Bees - classification</subject><subject>Bees - genetics</subject><subject>Bees - growth & development</subject><subject>Bees - microbiology</subject><subject>Biodegradation</subject><subject>Endangered species</subject><subject>Environmental degradation</subject><subject>Escherichia coli</subject><subject>Escherichia coli Infections - genetics</subject><subject>Escherichia coli Infections - microbiology</subject><subject>Fat Body</subject><subject>Female</subject><subject>Gene expression</subject><subject>Gene Expression - drug effects</subject><subject>Genes, Essential</subject><subject>Glyceraldehyde-3-phosphate dehydrogenase</subject><subject>Head</subject><subject>Humanities and Social Sciences</subject><subject>Larva - genetics</subject><subject>Male</subject><subject>multidisciplinary</subject><subject>Ovary</subject><subject>Pesticides</subject><subject>Pesticides - pharmacology</subject><subject>Pollinators</subject><subject>Polymerase chain reaction</subject><subject>Pupa - genetics</subject><subject>Real-Time Polymerase Chain Reaction - methods</subject><subject>Reverse transcription</subject><subject>Science</subject><subject>Science (multidisciplinary)</subject><subject>Sex</subject><issn>2045-2322</issn><issn>2045-2322</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2019</creationdate><recordtype>article</recordtype><sourceid>C6C</sourceid><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><recordid>eNp9kc1u1DAUhSMEolXpC7BAlti0i4D_Y3eBVI36g1QEQrC2nORm6ipjZ2xn1HkbHhVPp5SywQv7Wv7uOdY9VfWW4A8EM_UxcSK0qjHRtWCC8xq_qA4p5qKmjNKXz-qD6jilO1yWoJoT_bo6YKRpJBP6sPp1sbHjbLMLHoUBRRgggu8ALcFDQkOIDxWC-ylCSjvMejtuk0uo3RbejnV2K0Dr2frsclHaAPq2-I5O1mU_Rc6jfBsBUMrOL8eigdrdbYLOFYOT6-0KfJgyRHuGzifXWzhDX2B0U_DOu9M31avBjgmOH8-j6uflxY_FdX3z9erz4vym7gTHudZYiZ41VrR93xDL2lbKptMgqQAp-34YWiap7pu-07pTHDjjCtigqFID0YQdVZ_2utPcrqDvwOdoRzNFt7Jxa4J15t8X727NMmyMVIowxovA-0eBGNYzpGzuwhzLrJKhrDgoSbAoFN1TXQwplXE_ORBsdsGafbCmBGsegjW4NL17_renlj8xFoDtgVSe_BLiX-__yP4Giv-xXQ</recordid><startdate>20191127</startdate><enddate>20191127</enddate><creator>Freitas, Flávia C. P.</creator><creator>Depintor, Thiago S.</creator><creator>Agostini, Lucas T.</creator><creator>Luna-Lucena, Danielle</creator><creator>Nunes, Francis M. F.</creator><creator>Bitondi, Márcia M. G.</creator><creator>Simões, Zilá L. 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P. ; Depintor, Thiago S. ; Agostini, Lucas T. ; Luna-Lucena, Danielle ; Nunes, Francis M. F. ; Bitondi, Márcia M. G. ; Simões, Zilá L. 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P.</creatorcontrib><creatorcontrib>Depintor, Thiago S.</creatorcontrib><creatorcontrib>Agostini, Lucas T.</creatorcontrib><creatorcontrib>Luna-Lucena, Danielle</creatorcontrib><creatorcontrib>Nunes, Francis M. F.</creatorcontrib><creatorcontrib>Bitondi, Márcia M. G.</creatorcontrib><creatorcontrib>Simões, Zilá L. P.</creatorcontrib><creatorcontrib>Lourenço, Anete P.</creatorcontrib><collection>Springer Nature OA Free Journals</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Biology Database (Alumni Edition)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Science Database (Alumni Edition)</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest One Sustainability</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Science Database</collection><collection>Biological Science Database</collection><collection>Publicly Available Content Database</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central Basic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Scientific reports</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Freitas, Flávia C. P.</au><au>Depintor, Thiago S.</au><au>Agostini, Lucas T.</au><au>Luna-Lucena, Danielle</au><au>Nunes, Francis M. F.</au><au>Bitondi, Márcia M. G.</au><au>Simões, Zilá L. P.</au><au>Lourenço, Anete P.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Evaluation of reference genes for gene expression analysis by real-time quantitative PCR (qPCR) in three stingless bee species (Hymenoptera: Apidae: Meliponini)</atitle><jtitle>Scientific reports</jtitle><stitle>Sci Rep</stitle><addtitle>Sci Rep</addtitle><date>2019-11-27</date><risdate>2019</risdate><volume>9</volume><issue>1</issue><spage>17692</spage><epage>13</epage><pages>17692-13</pages><artnum>17692</artnum><issn>2045-2322</issn><eissn>2045-2322</eissn><abstract>Stingless bees are generalist pollinators distributed through the pantropical region. There is growing evidence that their wild populations are experiencing substantial decline in response to habitat degradation and pesticides. Policies for conservation of endangered species will benefit from studies focusing on genetic and molecular aspects of their development and behavior. The most common method for looking at gene expression is real-time quantitative polymerase chain reaction preceded by reverse transcription (RT-qPCR) of the mRNA of interest. This method requires the identification of reliable reference genes to correctly estimate fluctuations in transcript levels. To contribute to molecular studies on stingless bees, we used
Frieseomelitta varia
,
Melipona quadrifasciata
, and
Scaptotrigona bipunctata
species to test the expression stability of eight reference genes (
act
,
ef1-α
,
gapdh
,
rpl32
,
rps5
,
rps18
,
tbp
, and
tbp-af
) in RT-qPCR procedures in five physiological and experimental conditions (development, sex, tissues, bacteria injection, and pesticide exposure). In general, the
rpl32
,
rps5
and
rps18
ribosomal protein genes and
tpb-af
gene showed the highest stability, thus being identified as suitable reference genes for the three stingless bee species and defined conditions. Our results also emphasized the need to evaluate the stability of candidate genes for any designed experimental condition and stingless bee species.</abstract><cop>London</cop><pub>Nature Publishing Group UK</pub><pmid>31776359</pmid><doi>10.1038/s41598-019-53544-0</doi><tpages>13</tpages><orcidid>https://orcid.org/0000-0001-6929-7277</orcidid><orcidid>https://orcid.org/0000-0002-3162-4890</orcidid><oa>free_for_read</oa></addata></record> |
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source | MEDLINE; Nature Free; DOAJ Directory of Open Access Journals; EZB-FREE-00999 freely available EZB journals; PubMed Central; Alma/SFX Local Collection; Free Full-Text Journals in Chemistry; Springer Nature OA Free Journals |
subjects | 38/77 631/1647/2017 631/337/572 Animals Bees Bees - classification Bees - genetics Bees - growth & development Bees - microbiology Biodegradation Endangered species Environmental degradation Escherichia coli Escherichia coli Infections - genetics Escherichia coli Infections - microbiology Fat Body Female Gene expression Gene Expression - drug effects Genes, Essential Glyceraldehyde-3-phosphate dehydrogenase Head Humanities and Social Sciences Larva - genetics Male multidisciplinary Ovary Pesticides Pesticides - pharmacology Pollinators Polymerase chain reaction Pupa - genetics Real-Time Polymerase Chain Reaction - methods Reverse transcription Science Science (multidisciplinary) Sex |
title | Evaluation of reference genes for gene expression analysis by real-time quantitative PCR (qPCR) in three stingless bee species (Hymenoptera: Apidae: Meliponini) |
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