Selection of reference genes for quantitative PCR analysis in Citrus aurantifolia during phytoplasma infection
Quantifying gene expression is essential in most functional genomics experiments. For quantitative PCR (qPCR) assays, reproducible results are dependent on the correct choice of the reference genes for data normalization. To date, screenings for candidate reference genes suitable for expression stud...
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| Veröffentlicht in: | Tropical Plant Pathology 2018-10, Vol.43 (5), p.402-412 |
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| creator | Alves, Murilo S. Al-Sadi, Abdullah M. Carvalho, Claudine M. |
| description | Quantifying gene expression is essential in most functional genomics experiments. For quantitative PCR (qPCR) assays, reproducible results are dependent on the correct choice of the reference genes for data normalization. To date, screenings for candidate reference genes suitable for expression studies on plant-phytoplasma interactions in plants have not been reported. In the present study, we analyzed the expression patterns of 14 genes in midrib samples of
C. aurantifolia
plants infected with a ‘
Candidatus
Phytoplasma aurantifolia’ strain. Using GeNormPlus, NormFinder and BestKeeper algorithms, as well as testing relative expression by REST2009 software, the expression stability of several “classical” reference genes, such as
GAPDH
,
CYCLOPHILIN
and
18S rRNA
, and of newly identified candidates, was assessed. Our results showed similar performance among GeNormPlus, NormFinder and BestKeeper in evaluating the suitability of reference genes, with few differences among the top five genes. Furthermore, our data showed that some of the widely used reference genes for relative expression normalization in plants, including citrus lineages, were not the most stably expressed transcripts. In conclusion, we provide a list of validated reference genes and their relative primer sequences, usable to conduct reliable qPCR experiments in
C. aurantifolia
during phytoplasma infection. |
| doi_str_mv | 10.1007/s40858-018-0224-2 |
| format | Article |
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C. aurantifolia
plants infected with a ‘
Candidatus
Phytoplasma aurantifolia’ strain. Using GeNormPlus, NormFinder and BestKeeper algorithms, as well as testing relative expression by REST2009 software, the expression stability of several “classical” reference genes, such as
GAPDH
,
CYCLOPHILIN
and
18S rRNA
, and of newly identified candidates, was assessed. Our results showed similar performance among GeNormPlus, NormFinder and BestKeeper in evaluating the suitability of reference genes, with few differences among the top five genes. Furthermore, our data showed that some of the widely used reference genes for relative expression normalization in plants, including citrus lineages, were not the most stably expressed transcripts. In conclusion, we provide a list of validated reference genes and their relative primer sequences, usable to conduct reliable qPCR experiments in
C. aurantifolia
during phytoplasma infection.</description><identifier>ISSN: 1983-2052</identifier><identifier>ISSN: 1982-5676</identifier><identifier>EISSN: 1983-2052</identifier><identifier>DOI: 10.1007/s40858-018-0224-2</identifier><language>eng</language><publisher>Cham: Springer International Publishing</publisher><subject>Biomedical and Life Sciences ; Candidatus Phytoplasma aurantifolia ; Citrus ; Citrus aurantifolia ; Citrus fruits ; Gene expression ; Gene sequencing ; Genes ; Genomics ; Glyceraldehyde-3-phosphate dehydrogenase ; Health aspects ; Infections ; Life Sciences ; Original Article ; Phytoplasma ; Plant Pathology ; Polymerase chain reaction ; RNA ; rRNA 18S</subject><ispartof>Tropical Plant Pathology, 2018-10, Vol.43 (5), p.402-412</ispartof><rights>Sociedade Brasileira de Fitopatologia 2018</rights><rights>COPYRIGHT 2018 Springer</rights><rights>Copyright Springer Science & Business Media 2018</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c398t-7ee18773f5c9b5cdca06d7d07fa65ab31a6532f4ac788ae56e07dfd0b21a3b2f3</citedby><cites>FETCH-LOGICAL-c398t-7ee18773f5c9b5cdca06d7d07fa65ab31a6532f4ac788ae56e07dfd0b21a3b2f3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s40858-018-0224-2$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s40858-018-0224-2$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>315,782,786,27931,27932,41495,42564,51326</link.rule.ids></links><search><creatorcontrib>Alves, Murilo S.</creatorcontrib><creatorcontrib>Al-Sadi, Abdullah M.</creatorcontrib><creatorcontrib>Carvalho, Claudine M.</creatorcontrib><title>Selection of reference genes for quantitative PCR analysis in Citrus aurantifolia during phytoplasma infection</title><title>Tropical Plant Pathology</title><addtitle>Trop. plant pathol</addtitle><description>Quantifying gene expression is essential in most functional genomics experiments. For quantitative PCR (qPCR) assays, reproducible results are dependent on the correct choice of the reference genes for data normalization. To date, screenings for candidate reference genes suitable for expression studies on plant-phytoplasma interactions in plants have not been reported. In the present study, we analyzed the expression patterns of 14 genes in midrib samples of
C. aurantifolia
plants infected with a ‘
Candidatus
Phytoplasma aurantifolia’ strain. Using GeNormPlus, NormFinder and BestKeeper algorithms, as well as testing relative expression by REST2009 software, the expression stability of several “classical” reference genes, such as
GAPDH
,
CYCLOPHILIN
and
18S rRNA
, and of newly identified candidates, was assessed. Our results showed similar performance among GeNormPlus, NormFinder and BestKeeper in evaluating the suitability of reference genes, with few differences among the top five genes. Furthermore, our data showed that some of the widely used reference genes for relative expression normalization in plants, including citrus lineages, were not the most stably expressed transcripts. In conclusion, we provide a list of validated reference genes and their relative primer sequences, usable to conduct reliable qPCR experiments in
C. aurantifolia
during phytoplasma infection.</description><subject>Biomedical and Life Sciences</subject><subject>Candidatus Phytoplasma aurantifolia</subject><subject>Citrus</subject><subject>Citrus aurantifolia</subject><subject>Citrus fruits</subject><subject>Gene expression</subject><subject>Gene sequencing</subject><subject>Genes</subject><subject>Genomics</subject><subject>Glyceraldehyde-3-phosphate dehydrogenase</subject><subject>Health aspects</subject><subject>Infections</subject><subject>Life Sciences</subject><subject>Original Article</subject><subject>Phytoplasma</subject><subject>Plant Pathology</subject><subject>Polymerase chain reaction</subject><subject>RNA</subject><subject>rRNA 18S</subject><issn>1983-2052</issn><issn>1982-5676</issn><issn>1983-2052</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2018</creationdate><recordtype>article</recordtype><recordid>eNp1kVtLJDEQhRtxwcvuD_At4HO7lfQlmUcZdBUERXefQ026MkZ6kjZJL8y_3wwtrC8SQhXF-ZJDnaq64HDFAeTP1ILqVA28XCHaWhxVp3ylmlpAJ44_9SfVWUpvAL1Y9eq08i80kskueBYsi2QpkjfEtuQpMRsie5_RZ5cxu7_EntbPDD2O--QSc56tXY5zYjjHg8iG0SEb5uj8lk2v-xymEdMOi9Iun3yvvlkcE_34qOfVn9ub3-u7-uHx1_36-qE2zUrlWhJxJWVjO7PadGYwCP0gB5AW-w43DS-lEbZFI5VC6noCOdgBNoJjsxG2Oa8ul3enGN5nSlm_hTkW40kLzrumlwqgqK4W1RZH0sVkyBFNOQPtnAmerCvzawmyBSnavgB8AUwMKZVt6Sm6Hca95qAPOeglB11y0IcctCiMWJg0HfZC8b-Vr6F_5GWNCg</recordid><startdate>20181001</startdate><enddate>20181001</enddate><creator>Alves, Murilo S.</creator><creator>Al-Sadi, Abdullah M.</creator><creator>Carvalho, Claudine M.</creator><general>Springer International Publishing</general><general>Springer</general><general>Springer Nature B.V</general><scope>AAYXX</scope><scope>CITATION</scope><scope>IAO</scope></search><sort><creationdate>20181001</creationdate><title>Selection of reference genes for quantitative PCR analysis in Citrus aurantifolia during phytoplasma infection</title><author>Alves, Murilo S. ; Al-Sadi, Abdullah M. ; Carvalho, Claudine M.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c398t-7ee18773f5c9b5cdca06d7d07fa65ab31a6532f4ac788ae56e07dfd0b21a3b2f3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2018</creationdate><topic>Biomedical and Life Sciences</topic><topic>Candidatus Phytoplasma aurantifolia</topic><topic>Citrus</topic><topic>Citrus aurantifolia</topic><topic>Citrus fruits</topic><topic>Gene expression</topic><topic>Gene sequencing</topic><topic>Genes</topic><topic>Genomics</topic><topic>Glyceraldehyde-3-phosphate dehydrogenase</topic><topic>Health aspects</topic><topic>Infections</topic><topic>Life Sciences</topic><topic>Original Article</topic><topic>Phytoplasma</topic><topic>Plant Pathology</topic><topic>Polymerase chain reaction</topic><topic>RNA</topic><topic>rRNA 18S</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Alves, Murilo S.</creatorcontrib><creatorcontrib>Al-Sadi, Abdullah M.</creatorcontrib><creatorcontrib>Carvalho, Claudine M.</creatorcontrib><collection>CrossRef</collection><collection>Gale Academic OneFile</collection><jtitle>Tropical Plant Pathology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Alves, Murilo S.</au><au>Al-Sadi, Abdullah M.</au><au>Carvalho, Claudine M.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Selection of reference genes for quantitative PCR analysis in Citrus aurantifolia during phytoplasma infection</atitle><jtitle>Tropical Plant Pathology</jtitle><stitle>Trop. plant pathol</stitle><date>2018-10-01</date><risdate>2018</risdate><volume>43</volume><issue>5</issue><spage>402</spage><epage>412</epage><pages>402-412</pages><issn>1983-2052</issn><issn>1982-5676</issn><eissn>1983-2052</eissn><abstract>Quantifying gene expression is essential in most functional genomics experiments. For quantitative PCR (qPCR) assays, reproducible results are dependent on the correct choice of the reference genes for data normalization. To date, screenings for candidate reference genes suitable for expression studies on plant-phytoplasma interactions in plants have not been reported. In the present study, we analyzed the expression patterns of 14 genes in midrib samples of
C. aurantifolia
plants infected with a ‘
Candidatus
Phytoplasma aurantifolia’ strain. Using GeNormPlus, NormFinder and BestKeeper algorithms, as well as testing relative expression by REST2009 software, the expression stability of several “classical” reference genes, such as
GAPDH
,
CYCLOPHILIN
and
18S rRNA
, and of newly identified candidates, was assessed. Our results showed similar performance among GeNormPlus, NormFinder and BestKeeper in evaluating the suitability of reference genes, with few differences among the top five genes. Furthermore, our data showed that some of the widely used reference genes for relative expression normalization in plants, including citrus lineages, were not the most stably expressed transcripts. In conclusion, we provide a list of validated reference genes and their relative primer sequences, usable to conduct reliable qPCR experiments in
C. aurantifolia
during phytoplasma infection.</abstract><cop>Cham</cop><pub>Springer International Publishing</pub><doi>10.1007/s40858-018-0224-2</doi><tpages>11</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | Tropical Plant Pathology, 2018-10, Vol.43 (5), p.402-412 |
| issn | 1983-2052 1982-5676 1983-2052 |
| language | eng |
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| subjects | Biomedical and Life Sciences Candidatus Phytoplasma aurantifolia Citrus Citrus aurantifolia Citrus fruits Gene expression Gene sequencing Genes Genomics Glyceraldehyde-3-phosphate dehydrogenase Health aspects Infections Life Sciences Original Article Phytoplasma Plant Pathology Polymerase chain reaction RNA rRNA 18S |
| title | Selection of reference genes for quantitative PCR analysis in Citrus aurantifolia during phytoplasma infection |
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