Selection of suitable reference genes for normalization of quantitative RT-PCR (RT-qPCR) expression data across twelve tissues of riverine buffaloes (Bubalus bubalis)
Identifieur interne : 000113 ( Pmc/Checkpoint ); précédent : 000112; suivant : 000114Selection of suitable reference genes for normalization of quantitative RT-PCR (RT-qPCR) expression data across twelve tissues of riverine buffaloes (Bubalus bubalis)
Auteurs : Ramneek Kaur [Inde] ; Monika Sodhi [Inde] ; Ankita Sharma [Inde] ; Vijay Lakshmi Sharma [Inde] ; Preeti Verma [Inde] ; Shelesh Kumar Swami [Inde] ; Parvesh Kumari [Inde] ; Manishi Mukesh [Inde]Source :
- PLoS ONE [ 1932-6203 ] ; 2018.
Abstract
Selection of reference genes has become an integral step in any real time quantitative PCR (RT-qPCR) based expression studies. The importance of this study stems from the fact that riverine buffaloes are major dairy species of Indian sub-continent and the information generated here will be of great interest to the investigators engaged in functional genomic studies of this important livestock species. In this study, an effort was made to evaluate a panel of 10 candidate reference genes (glyceraldehyde 3-phosphate dehydrogenase (
Url:
DOI: 10.1371/journal.pone.0191558
PubMed: 29509770
PubMed Central: 5839537
Affiliations:
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Selection of suitable reference genes for normalization of quantitative RT-PCR (RT-qPCR) expression data across twelve tissues of riverine buffaloes (<italic>Bubalus bubalis</italic>)</title><author><name sortKey="Kaur, Ramneek" sort="Kaur, Ramneek" uniqKey="Kaur R" first="Ramneek" last="Kaur">Ramneek Kaur</name><affiliation wicri:level="1"><nlm:aff id="aff001"><addr-line>Department of Animal Biotechnology, ICAR-National Bureau of Animal Genetic Resources, Karnal, Haryana, India</addr-line></nlm:aff><country xml:lang="fr">Inde</country><wicri:regionArea>Department of Animal Biotechnology, ICAR-National Bureau of Animal Genetic Resources, Karnal, Haryana</wicri:regionArea><wicri:noRegion>Haryana</wicri:noRegion></affiliation><affiliation wicri:level="1"><nlm:aff id="aff002"><addr-line>Department of Zoology, Panjab University, Chandigarh, India</addr-line></nlm:aff><country xml:lang="fr">Inde</country><wicri:regionArea>Department of Zoology, Panjab University, Chandigarh</wicri:regionArea><wicri:noRegion>Chandigarh</wicri:noRegion></affiliation></author><author><name sortKey="Sodhi, Monika" sort="Sodhi, Monika" uniqKey="Sodhi M" first="Monika" last="Sodhi">Monika Sodhi</name><affiliation wicri:level="1"><nlm:aff id="aff001"><addr-line>Department of Animal Biotechnology, ICAR-National Bureau of Animal Genetic Resources, Karnal, Haryana, India</addr-line></nlm:aff><country xml:lang="fr">Inde</country><wicri:regionArea>Department of Animal Biotechnology, ICAR-National Bureau of Animal Genetic Resources, Karnal, Haryana</wicri:regionArea><wicri:noRegion>Haryana</wicri:noRegion></affiliation></author><author><name sortKey="Sharma, Ankita" sort="Sharma, Ankita" uniqKey="Sharma A" first="Ankita" last="Sharma">Ankita Sharma</name><affiliation wicri:level="1"><nlm:aff id="aff001"><addr-line>Department of Animal Biotechnology, ICAR-National Bureau of Animal Genetic Resources, Karnal, Haryana, India</addr-line></nlm:aff><country xml:lang="fr">Inde</country><wicri:regionArea>Department of Animal Biotechnology, ICAR-National Bureau of Animal Genetic Resources, Karnal, Haryana</wicri:regionArea><wicri:noRegion>Haryana</wicri:noRegion></affiliation></author><author><name sortKey="Sharma, Vijay Lakshmi" sort="Sharma, Vijay Lakshmi" uniqKey="Sharma V" first="Vijay Lakshmi" last="Sharma">Vijay Lakshmi Sharma</name><affiliation wicri:level="1"><nlm:aff id="aff002"><addr-line>Department of Zoology, Panjab University, Chandigarh, India</addr-line></nlm:aff><country xml:lang="fr">Inde</country><wicri:regionArea>Department of Zoology, Panjab University, Chandigarh</wicri:regionArea><wicri:noRegion>Chandigarh</wicri:noRegion></affiliation></author><author><name sortKey="Verma, Preeti" sort="Verma, Preeti" uniqKey="Verma P" first="Preeti" last="Verma">Preeti Verma</name><affiliation wicri:level="1"><nlm:aff id="aff001"><addr-line>Department of Animal Biotechnology, ICAR-National Bureau of Animal Genetic Resources, Karnal, Haryana, India</addr-line></nlm:aff><country xml:lang="fr">Inde</country><wicri:regionArea>Department of Animal Biotechnology, ICAR-National Bureau of Animal Genetic Resources, Karnal, Haryana</wicri:regionArea><wicri:noRegion>Haryana</wicri:noRegion></affiliation></author><author><name sortKey="Swami, Shelesh Kumar" sort="Swami, Shelesh Kumar" uniqKey="Swami S" first="Shelesh Kumar" last="Swami">Shelesh Kumar Swami</name><affiliation wicri:level="1"><nlm:aff id="aff001"><addr-line>Department of Animal Biotechnology, ICAR-National Bureau of Animal Genetic Resources, Karnal, Haryana, India</addr-line></nlm:aff><country xml:lang="fr">Inde</country><wicri:regionArea>Department of Animal Biotechnology, ICAR-National Bureau of Animal Genetic Resources, Karnal, Haryana</wicri:regionArea><wicri:noRegion>Haryana</wicri:noRegion></affiliation></author><author><name sortKey="Kumari, Parvesh" sort="Kumari, Parvesh" uniqKey="Kumari P" first="Parvesh" last="Kumari">Parvesh Kumari</name><affiliation wicri:level="1"><nlm:aff id="aff001"><addr-line>Department of Animal Biotechnology, ICAR-National Bureau of Animal Genetic Resources, Karnal, Haryana, India</addr-line></nlm:aff><country xml:lang="fr">Inde</country><wicri:regionArea>Department of Animal Biotechnology, ICAR-National Bureau of Animal Genetic Resources, Karnal, Haryana</wicri:regionArea><wicri:noRegion>Haryana</wicri:noRegion></affiliation></author><author><name sortKey="Mukesh, Manishi" sort="Mukesh, Manishi" uniqKey="Mukesh M" first="Manishi" last="Mukesh">Manishi Mukesh</name><affiliation wicri:level="1"><nlm:aff id="aff001"><addr-line>Department of Animal Biotechnology, ICAR-National Bureau of Animal Genetic Resources, Karnal, Haryana, India</addr-line></nlm:aff><country xml:lang="fr">Inde</country><wicri:regionArea>Department of Animal Biotechnology, ICAR-National Bureau of Animal Genetic Resources, Karnal, Haryana</wicri:regionArea><wicri:noRegion>Haryana</wicri:noRegion></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">29509770</idno><idno type="pmc">5839537</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5839537</idno><idno type="RBID">PMC:5839537</idno><idno type="doi">10.1371/journal.pone.0191558</idno><date when="2018">2018</date><idno type="wicri:Area/Pmc/Corpus">000687</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000687</idno><idno type="wicri:Area/Pmc/Curation">000687</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Curation">000687</idno><idno type="wicri:Area/Pmc/Checkpoint">000113</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Checkpoint">000113</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Selection of suitable reference genes for normalization of quantitative RT-PCR (RT-qPCR) expression data across twelve tissues of riverine buffaloes (<italic>Bubalus bubalis</italic>)</title><author><name sortKey="Kaur, Ramneek" sort="Kaur, Ramneek" uniqKey="Kaur R" first="Ramneek" last="Kaur">Ramneek Kaur</name><affiliation wicri:level="1"><nlm:aff id="aff001"><addr-line>Department of Animal Biotechnology, ICAR-National Bureau of Animal Genetic Resources, Karnal, Haryana, India</addr-line></nlm:aff><country xml:lang="fr">Inde</country><wicri:regionArea>Department of Animal Biotechnology, ICAR-National Bureau of Animal Genetic Resources, Karnal, Haryana</wicri:regionArea><wicri:noRegion>Haryana</wicri:noRegion></affiliation><affiliation wicri:level="1"><nlm:aff id="aff002"><addr-line>Department of Zoology, Panjab University, Chandigarh, India</addr-line></nlm:aff><country xml:lang="fr">Inde</country><wicri:regionArea>Department of Zoology, Panjab University, Chandigarh</wicri:regionArea><wicri:noRegion>Chandigarh</wicri:noRegion></affiliation></author><author><name sortKey="Sodhi, Monika" sort="Sodhi, Monika" uniqKey="Sodhi M" first="Monika" last="Sodhi">Monika Sodhi</name><affiliation wicri:level="1"><nlm:aff id="aff001"><addr-line>Department of Animal Biotechnology, ICAR-National Bureau of Animal Genetic Resources, Karnal, Haryana, India</addr-line></nlm:aff><country xml:lang="fr">Inde</country><wicri:regionArea>Department of Animal Biotechnology, ICAR-National Bureau of Animal Genetic Resources, Karnal, Haryana</wicri:regionArea><wicri:noRegion>Haryana</wicri:noRegion></affiliation></author><author><name sortKey="Sharma, Ankita" sort="Sharma, Ankita" uniqKey="Sharma A" first="Ankita" last="Sharma">Ankita Sharma</name><affiliation wicri:level="1"><nlm:aff id="aff001"><addr-line>Department of Animal Biotechnology, ICAR-National Bureau of Animal Genetic Resources, Karnal, Haryana, India</addr-line></nlm:aff><country xml:lang="fr">Inde</country><wicri:regionArea>Department of Animal Biotechnology, ICAR-National Bureau of Animal Genetic Resources, Karnal, Haryana</wicri:regionArea><wicri:noRegion>Haryana</wicri:noRegion></affiliation></author><author><name sortKey="Sharma, Vijay Lakshmi" sort="Sharma, Vijay Lakshmi" uniqKey="Sharma V" first="Vijay Lakshmi" last="Sharma">Vijay Lakshmi Sharma</name><affiliation wicri:level="1"><nlm:aff id="aff002"><addr-line>Department of Zoology, Panjab University, Chandigarh, India</addr-line></nlm:aff><country xml:lang="fr">Inde</country><wicri:regionArea>Department of Zoology, Panjab University, Chandigarh</wicri:regionArea><wicri:noRegion>Chandigarh</wicri:noRegion></affiliation></author><author><name sortKey="Verma, Preeti" sort="Verma, Preeti" uniqKey="Verma P" first="Preeti" last="Verma">Preeti Verma</name><affiliation wicri:level="1"><nlm:aff id="aff001"><addr-line>Department of Animal Biotechnology, ICAR-National Bureau of Animal Genetic Resources, Karnal, Haryana, India</addr-line></nlm:aff><country xml:lang="fr">Inde</country><wicri:regionArea>Department of Animal Biotechnology, ICAR-National Bureau of Animal Genetic Resources, Karnal, Haryana</wicri:regionArea><wicri:noRegion>Haryana</wicri:noRegion></affiliation></author><author><name sortKey="Swami, Shelesh Kumar" sort="Swami, Shelesh Kumar" uniqKey="Swami S" first="Shelesh Kumar" last="Swami">Shelesh Kumar Swami</name><affiliation wicri:level="1"><nlm:aff id="aff001"><addr-line>Department of Animal Biotechnology, ICAR-National Bureau of Animal Genetic Resources, Karnal, Haryana, India</addr-line></nlm:aff><country xml:lang="fr">Inde</country><wicri:regionArea>Department of Animal Biotechnology, ICAR-National Bureau of Animal Genetic Resources, Karnal, Haryana</wicri:regionArea><wicri:noRegion>Haryana</wicri:noRegion></affiliation></author><author><name sortKey="Kumari, Parvesh" sort="Kumari, Parvesh" uniqKey="Kumari P" first="Parvesh" last="Kumari">Parvesh Kumari</name><affiliation wicri:level="1"><nlm:aff id="aff001"><addr-line>Department of Animal Biotechnology, ICAR-National Bureau of Animal Genetic Resources, Karnal, Haryana, India</addr-line></nlm:aff><country xml:lang="fr">Inde</country><wicri:regionArea>Department of Animal Biotechnology, ICAR-National Bureau of Animal Genetic Resources, Karnal, Haryana</wicri:regionArea><wicri:noRegion>Haryana</wicri:noRegion></affiliation></author><author><name sortKey="Mukesh, Manishi" sort="Mukesh, Manishi" uniqKey="Mukesh M" first="Manishi" last="Mukesh">Manishi Mukesh</name><affiliation wicri:level="1"><nlm:aff id="aff001"><addr-line>Department of Animal Biotechnology, ICAR-National Bureau of Animal Genetic Resources, Karnal, Haryana, India</addr-line></nlm:aff><country xml:lang="fr">Inde</country><wicri:regionArea>Department of Animal Biotechnology, ICAR-National Bureau of Animal Genetic Resources, Karnal, Haryana</wicri:regionArea><wicri:noRegion>Haryana</wicri:noRegion></affiliation></author></analytic><series><title level="j">PLoS ONE</title><idno type="eISSN">1932-6203</idno><imprint><date when="2018">2018</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Selection of reference genes has become an integral step in any real time quantitative PCR (RT-qPCR) based expression studies. The importance of this study stems from the fact that riverine buffaloes are major dairy species of Indian sub-continent and the information generated here will be of great interest to the investigators engaged in functional genomic studies of this important livestock species. In this study, an effort was made to evaluate a panel of 10 candidate reference genes (glyceraldehyde 3-phosphate dehydrogenase (<italic>GAPDH)</italic>, beta- actin (<italic>ACTB</italic>), ubiquitously expressed transcript (<italic>UXT</italic>), ribosomal protein S15 (<italic>RPS15</italic>), ribosomal protein L-4 (<italic>RPL4</italic>), ribosomal protein S9 (<italic>RPS9</italic>), ribosomal protein S23 (<italic>RPS23</italic>), hydroxymethylbilane synthase (<italic>HMBS</italic>), β2 Microglobulin (<italic>β2M</italic>) and eukaryotic translation elongation factor 1 alpha 1 (<italic>EEF1A1</italic>) across 12 tissues (mammary gland, kidney, spleen, liver, heart, intestine, ovary, lung, muscle, brain, subcutaneous fat and testis) of riverine buffaloes. In addition to overall analysis, tissue wise evaluation of expression stability of individual RG was also performed. Three different algorithms provided in geNorm, NormFinder and BestKeeper softwares were used to evaluate the stability of 10 potential reference genes from different functional classes. The M-value given by geNorm ranged from 0.9797 (<italic>RPS9</italic> and <italic>UXT</italic>) to 1.7362 (<italic>RPS15</italic>). From the most stable to the least stable, genes were ranked as: <italic>UXT/RPS9> RPL4> RPS23> EEF1A1> ACTB> HMBS> GAPDH> B2M> RPS15</italic>. While NormFinder analysis ranked the genes as: <italic>UXT> RPS23> RPL4> RPS9> EEF1A1> HMBS> ACTB> β2M> GAPDH> RPS15</italic>. Based on the crossing point SD value and range of fold change expression, BestKeeper analysis ranked the genes as: <italic>RPS9> RPS23/UXT> RPL4> GAPDH> EEF1A1> ACTB> HMBS> β2M> RPS15</italic>. Overall the study has identified <italic>RPS23</italic>, <italic>RPS9</italic>, <italic>RPL4</italic> and <italic>UXT</italic> genes to be the most stable and appropriate RGs that could be utilized for normalization of transcriptional data in various tissues of buffaloes. This manuscript thus provide useful information on panel of reference genes that could be helpful for researchers conducting functional genomic studies in riverine buffaloes.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Livak, Kj" uniqKey="Livak K">KJ Livak</name></author><author><name sortKey="Schmittgen, Td" uniqKey="Schmittgen T">TD Schmittgen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bustin, S A" uniqKey="Bustin S">S.A Bustin</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Fleige, S" uniqKey="Fleige S">S Fleige</name></author><author><name sortKey="Pfaffl, Mw" uniqKey="Pfaffl M">MW Pfaffl</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lisowski, P" uniqKey="Lisowski P">P Lisowski</name></author><author><name sortKey="Pierzchala, M" uniqKey="Pierzchala M">M 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<front><journal-meta><journal-id journal-id-type="nlm-ta">PLoS One</journal-id><journal-id journal-id-type="iso-abbrev">PLoS ONE</journal-id><journal-id journal-id-type="publisher-id">plos</journal-id><journal-id journal-id-type="pmc">plosone</journal-id><journal-title-group><journal-title>PLoS ONE</journal-title></journal-title-group><issn pub-type="epub">1932-6203</issn><publisher><publisher-name>Public Library of Science</publisher-name><publisher-loc>San Francisco, CA USA</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">29509770</article-id><article-id pub-id-type="pmc">5839537</article-id><article-id pub-id-type="doi">10.1371/journal.pone.0191558</article-id><article-id pub-id-type="publisher-id">PONE-D-16-42801</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Biology and Life Sciences</subject><subj-group><subject>Organisms</subject><subj-group><subject>Eukaryota</subject><subj-group><subject>Animals</subject><subj-group><subject>Vertebrates</subject><subj-group><subject>Amniotes</subject><subj-group><subject>Mammals</subject><subj-group><subject>Bovines</subject><subj-group><subject>Buffaloes</subject></subj-group></subj-group></subj-group></subj-group></subj-group></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Biology and Life Sciences</subject><subj-group><subject>Genetics</subject><subj-group><subject>Gene Expression</subject></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Research and analysis methods</subject><subj-group><subject>Extraction techniques</subject><subj-group><subject>RNA extraction</subject></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Biology and Life Sciences</subject><subj-group><subject>Molecular Biology</subject><subj-group><subject>Molecular Biology Techniques</subject><subj-group><subject>Artificial Gene Amplification and Extension</subject><subj-group><subject>Polymerase Chain Reaction</subject></subj-group></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Research and Analysis Methods</subject><subj-group><subject>Molecular Biology Techniques</subject><subj-group><subject>Artificial Gene Amplification and Extension</subject><subj-group><subject>Polymerase Chain Reaction</subject></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Biology and Life Sciences</subject><subj-group><subject>Genetics</subject><subj-group><subject>Gene Expression</subject><subj-group><subject>Gene Regulation</subject><subj-group><subject>Elongation Factors</subject></subj-group></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Biology and Life Sciences</subject><subj-group><subject>Biochemistry</subject><subj-group><subject>Proteins</subject><subj-group><subject>Regulatory Proteins</subject><subj-group><subject>Elongation Factors</subject></subj-group></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Biology and Life Sciences</subject><subj-group><subject>Anatomy</subject><subj-group><subject>Digestive System</subject><subj-group><subject>Gastrointestinal Tract</subject></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Medicine and Health Sciences</subject><subj-group><subject>Anatomy</subject><subj-group><subject>Digestive System</subject><subj-group><subject>Gastrointestinal Tract</subject></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Biology and Life Sciences</subject><subj-group><subject>Biochemistry</subject><subj-group><subject>Lipids</subject><subj-group><subject>Fats</subject></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Research and analysis methods</subject><subj-group><subject>Chemical synthesis</subject><subj-group><subject>Biosynthetic techniques</subject><subj-group><subject>Nucleic acid synthesis</subject><subj-group><subject>RNA synthesis</subject></subj-group></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Biology and life sciences</subject><subj-group><subject>Biochemistry</subject><subj-group><subject>Nucleic acids</subject><subj-group><subject>RNA</subject><subj-group><subject>RNA synthesis</subject></subj-group></subj-group></subj-group></subj-group></subj-group></article-categories><title-group><article-title>Selection of suitable reference genes for normalization of quantitative RT-PCR (RT-qPCR) expression data across twelve tissues of riverine buffaloes (<italic>Bubalus bubalis</italic>)</article-title><alt-title alt-title-type="running-head">Reference genes for qPCR studies across tissues of riverine buffaloes</alt-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Kaur</surname><given-names>Ramneek</given-names></name><role content-type="http://credit.casrai.org/">Data curation</role><role content-type="http://credit.casrai.org/">Formal analysis</role><role content-type="http://credit.casrai.org/">Writing – original draft</role><role content-type="http://credit.casrai.org/">Writing – review & editing</role><xref ref-type="aff" rid="aff001"><sup>1</sup></xref><xref ref-type="aff" rid="aff002"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Sodhi</surname><given-names>Monika</given-names></name><role content-type="http://credit.casrai.org/">Conceptualization</role><role content-type="http://credit.casrai.org/">Resources</role><role content-type="http://credit.casrai.org/">Writing – original draft</role><role content-type="http://credit.casrai.org/">Writing – review & editing</role><xref ref-type="aff" rid="aff001"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Sharma</surname><given-names>Ankita</given-names></name><role content-type="http://credit.casrai.org/">Data curation</role><role content-type="http://credit.casrai.org/">Formal analysis</role><role content-type="http://credit.casrai.org/">Writing – original draft</role><role content-type="http://credit.casrai.org/">Writing – review & editing</role><xref ref-type="aff" rid="aff001"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Sharma</surname><given-names>Vijay Lakshmi</given-names></name><role content-type="http://credit.casrai.org/">Conceptualization</role><role content-type="http://credit.casrai.org/">Resources</role><xref ref-type="aff" rid="aff002"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Verma</surname><given-names>Preeti</given-names></name><role content-type="http://credit.casrai.org/">Data curation</role><xref ref-type="aff" rid="aff001"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Swami</surname><given-names>Shelesh Kumar</given-names></name><role content-type="http://credit.casrai.org/">Data curation</role><xref ref-type="aff" rid="aff001"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Kumari</surname><given-names>Parvesh</given-names></name><role content-type="http://credit.casrai.org/">Data curation</role><role content-type="http://credit.casrai.org/">Resources</role><xref ref-type="aff" rid="aff001"><sup>1</sup></xref></contrib><contrib contrib-type="author"><contrib-id authenticated="true" contrib-id-type="orcid">http://orcid.org/0000-0001-8101-3863</contrib-id><name><surname>Mukesh</surname><given-names>Manishi</given-names></name><role content-type="http://credit.casrai.org/">Conceptualization</role><role content-type="http://credit.casrai.org/">Formal analysis</role><role content-type="http://credit.casrai.org/">Writing – original draft</role><role content-type="http://credit.casrai.org/">Writing – review & editing</role><xref ref-type="aff" rid="aff001"><sup>1</sup></xref><xref ref-type="corresp" rid="cor001">*</xref></contrib></contrib-group><aff id="aff001"><label>1</label><addr-line>Department of Animal Biotechnology, ICAR-National Bureau of Animal Genetic Resources, Karnal, Haryana, India</addr-line></aff><aff id="aff002"><label>2</label><addr-line>Department of Zoology, Panjab University, Chandigarh, India</addr-line></aff><contrib-group><contrib contrib-type="editor"><name><surname>Schönbach</surname><given-names>Christian</given-names></name><role>Editor</role><xref ref-type="aff" rid="edit1"></xref></contrib></contrib-group><aff id="edit1"><addr-line>Nazarbayev University, KAZAKHSTAN</addr-line></aff><author-notes><fn fn-type="COI-statement" id="coi001"><p><bold>Competing Interests: </bold>The authors have declared that no competing interests exist.</p></fn><corresp id="cor001">* E-mail: <email>mmukesh_26@hotmail.com</email>, <email>mmukesh26@gmail.com</email></corresp></author-notes><pub-date pub-type="epub"><day>6</day><month>3</month><year>2018</year></pub-date><pub-date pub-type="collection"><year>2018</year></pub-date><volume>13</volume><issue>3</issue><elocation-id>e0191558</elocation-id><history><date date-type="received"><day>27</day><month>10</month><year>2016</year></date><date date-type="accepted"><day>8</day><month>1</month><year>2018</year></date></history><permissions><copyright-statement>© 2018 Kaur et al</copyright-statement><copyright-year>2018</copyright-year><copyright-holder>Kaur et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This is an open access article distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="pone.0191558.pdf"></self-uri><abstract><p>Selection of reference genes has become an integral step in any real time quantitative PCR (RT-qPCR) based expression studies. The importance of this study stems from the fact that riverine buffaloes are major dairy species of Indian sub-continent and the information generated here will be of great interest to the investigators engaged in functional genomic studies of this important livestock species. In this study, an effort was made to evaluate a panel of 10 candidate reference genes (glyceraldehyde 3-phosphate dehydrogenase (<italic>GAPDH)</italic>, beta- actin (<italic>ACTB</italic>), ubiquitously expressed transcript (<italic>UXT</italic>), ribosomal protein S15 (<italic>RPS15</italic>), ribosomal protein L-4 (<italic>RPL4</italic>), ribosomal protein S9 (<italic>RPS9</italic>), ribosomal protein S23 (<italic>RPS23</italic>), hydroxymethylbilane synthase (<italic>HMBS</italic>), β2 Microglobulin (<italic>β2M</italic>) and eukaryotic translation elongation factor 1 alpha 1 (<italic>EEF1A1</italic>) across 12 tissues (mammary gland, kidney, spleen, liver, heart, intestine, ovary, lung, muscle, brain, subcutaneous fat and testis) of riverine buffaloes. In addition to overall analysis, tissue wise evaluation of expression stability of individual RG was also performed. Three different algorithms provided in geNorm, NormFinder and BestKeeper softwares were used to evaluate the stability of 10 potential reference genes from different functional classes. The M-value given by geNorm ranged from 0.9797 (<italic>RPS9</italic> and <italic>UXT</italic>) to 1.7362 (<italic>RPS15</italic>). From the most stable to the least stable, genes were ranked as: <italic>UXT/RPS9> RPL4> RPS23> EEF1A1> ACTB> HMBS> GAPDH> B2M> RPS15</italic>. While NormFinder analysis ranked the genes as: <italic>UXT> RPS23> RPL4> RPS9> EEF1A1> HMBS> ACTB> β2M> GAPDH> RPS15</italic>. Based on the crossing point SD value and range of fold change expression, BestKeeper analysis ranked the genes as: <italic>RPS9> RPS23/UXT> RPL4> GAPDH> EEF1A1> ACTB> HMBS> β2M> RPS15</italic>. Overall the study has identified <italic>RPS23</italic>, <italic>RPS9</italic>, <italic>RPL4</italic> and <italic>UXT</italic> genes to be the most stable and appropriate RGs that could be utilized for normalization of transcriptional data in various tissues of buffaloes. This manuscript thus provide useful information on panel of reference genes that could be helpful for researchers conducting functional genomic studies in riverine buffaloes.</p></abstract><funding-group><award-group id="award001"><funding-source><institution>National Fellow Scheme of Indian Council of Agricultural Research, New Delhi</institution></funding-source><award-id>27(3)/2010-HRD</award-id><principal-award-recipient><contrib-id authenticated="true" contrib-id-type="orcid">http://orcid.org/0000-0001-8101-3863</contrib-id><name><surname>Mukesh</surname><given-names>Manishi</given-names></name></principal-award-recipient></award-group><award-group id="award002"><funding-source><institution>Inspire Fellowship from DEpartment of Science and Technology, India</institution></funding-source><principal-award-recipient><name><surname>Kaur</surname><given-names>Ramneek</given-names></name></principal-award-recipient></award-group><funding-statement>This work was supported under National Fellow Scheme of Indian Council of Agricultural Research, New Delhi grant number 27(3)/2010-HRD, <ext-link ext-link-type="uri" xlink:href="http://www.icar.org.in/files/edu/Award-of-National-Fellow.pdf">http://www.icar.org.in/files/edu/Award-of-National-Fellow.pdf</ext-link>. MM, Principal Scientist and National Fellow, received the funding from ICAR. RK has received the inspire fellowship from Department of Science and Technology, Govt. of India.</funding-statement></funding-group><counts><fig-count count="9"></fig-count><table-count count="4"></table-count><page-count count="15"></page-count></counts><custom-meta-group><custom-meta id="data-availability"><meta-name>Data Availability</meta-name><meta-value>All relevant data are within the paper and its Supporting Information files.</meta-value></custom-meta></custom-meta-group></article-meta><notes><title>Data Availability</title><p>All relevant data are within the paper and its Supporting Information files.</p></notes></front></pmc><affiliations><list><country><li>Inde</li></country></list><tree><country name="Inde"><noRegion><name sortKey="Kaur, Ramneek" sort="Kaur, Ramneek" uniqKey="Kaur R" first="Ramneek" last="Kaur">Ramneek Kaur</name></noRegion><name sortKey="Kaur, Ramneek" sort="Kaur, Ramneek" uniqKey="Kaur R" first="Ramneek" last="Kaur">Ramneek Kaur</name><name sortKey="Kumari, Parvesh" sort="Kumari, Parvesh" uniqKey="Kumari P" first="Parvesh" last="Kumari">Parvesh Kumari</name><name sortKey="Mukesh, Manishi" sort="Mukesh, Manishi" uniqKey="Mukesh M" first="Manishi" last="Mukesh">Manishi Mukesh</name><name sortKey="Sharma, Ankita" sort="Sharma, Ankita" uniqKey="Sharma A" first="Ankita" last="Sharma">Ankita Sharma</name><name sortKey="Sharma, Vijay Lakshmi" sort="Sharma, Vijay Lakshmi" uniqKey="Sharma V" first="Vijay Lakshmi" last="Sharma">Vijay Lakshmi Sharma</name><name sortKey="Sodhi, Monika" sort="Sodhi, Monika" uniqKey="Sodhi M" first="Monika" last="Sodhi">Monika Sodhi</name><name sortKey="Swami, Shelesh Kumar" sort="Swami, Shelesh Kumar" uniqKey="Swami S" first="Shelesh Kumar" last="Swami">Shelesh Kumar Swami</name><name sortKey="Verma, Preeti" sort="Verma, Preeti" uniqKey="Verma P" first="Preeti" last="Verma">Preeti Verma</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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