Evaluation of putative reference genes for gene expression normalization in soybean by quantitative real-time RT-PCR
Real-time quantitative reverse transcription PCR (RT-qPCR) data needs to be normalized for its proper interpretation. Housekeeping genes are routinely employed for this purpose, but their expression level cannot be assumed to remain constant under all possible experimental conditions. Thus, a system...
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| description | Real-time quantitative reverse transcription PCR (RT-qPCR) data needs to be normalized for its proper interpretation. Housekeeping genes are routinely employed for this purpose, but their expression level cannot be assumed to remain constant under all possible experimental conditions. Thus, a systematic validation of reference genes is required to ensure proper normalization. For soybean, only a small number of validated reference genes are available to date.
A systematic comparison of 14 potential reference genes for soybean is presented. These included seven commonly used (ACT2, ACT11, TUB4, TUA5, CYP, UBQ10, EF1b) and seven new candidates (SKIP16, MTP, PEPKR1, HDC, TIP41, UKN1, UKN2). Expression stability was examined by RT-qPCR across 116 biological samples, representing tissues at various developmental stages, varied photoperiodic treatments, and a range of soybean cultivars. Expression of all 14 genes was variable to some extent, but that of SKIP16, UKN1 and UKN2 was overall the most stable. A combination of ACT11, UKN1 and UKN2 would be appropriate as a reference panel for normalizing gene expression data among different tissues, whereas the combination SKIP16, UKN1 and MTP was most suitable for developmental stages. ACT11, TUA5 and TIP41 were the most stably expressed when the photoperiod was altered, and TIP41, UKN1 and UKN2 when the light quality was changed. For six different cultivars in long day (LD) and short day (SD), their expression stability did not vary significantly with ACT11, UKN2 and TUB4 being the most stable genes. The relative gene expression level of GmFTL3, an ortholog of Arabidopsis FT (FLOWERING LOCUS T) was detected to validate the reference genes selected in this study.
None of the candidate reference genes was uniformly expressed across all experimental conditions, and the most suitable reference genes are conditional-, tissue-specific-, developmental-, and cultivar-dependent. Most of the new reference genes performed better than the conventional housekeeping genes. These results should guide the selection of reference genes for gene expression studies in soybean. |
| doi_str_mv | 10.1186/1471-2199-10-93 |
| format | Article |
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A systematic comparison of 14 potential reference genes for soybean is presented. These included seven commonly used (ACT2, ACT11, TUB4, TUA5, CYP, UBQ10, EF1b) and seven new candidates (SKIP16, MTP, PEPKR1, HDC, TIP41, UKN1, UKN2). Expression stability was examined by RT-qPCR across 116 biological samples, representing tissues at various developmental stages, varied photoperiodic treatments, and a range of soybean cultivars. Expression of all 14 genes was variable to some extent, but that of SKIP16, UKN1 and UKN2 was overall the most stable. A combination of ACT11, UKN1 and UKN2 would be appropriate as a reference panel for normalizing gene expression data among different tissues, whereas the combination SKIP16, UKN1 and MTP was most suitable for developmental stages. ACT11, TUA5 and TIP41 were the most stably expressed when the photoperiod was altered, and TIP41, UKN1 and UKN2 when the light quality was changed. For six different cultivars in long day (LD) and short day (SD), their expression stability did not vary significantly with ACT11, UKN2 and TUB4 being the most stable genes. The relative gene expression level of GmFTL3, an ortholog of Arabidopsis FT (FLOWERING LOCUS T) was detected to validate the reference genes selected in this study.
None of the candidate reference genes was uniformly expressed across all experimental conditions, and the most suitable reference genes are conditional-, tissue-specific-, developmental-, and cultivar-dependent. Most of the new reference genes performed better than the conventional housekeeping genes. These results should guide the selection of reference genes for gene expression studies in soybean.</description><identifier>ISSN: 1471-2199</identifier><identifier>EISSN: 1471-2199</identifier><identifier>DOI: 10.1186/1471-2199-10-93</identifier><identifier>PMID: 19785741</identifier><language>eng</language><publisher>England: BioMed Central Ltd</publisher><subject>Analysis ; Arabidopsis ; Crop science ; Cytochrome P-450 ; Data processing ; Gene expression ; Gene Expression Profiling ; Gene Expression Regulation, Plant ; Genes, Plant - genetics ; Genetic aspects ; Genomes ; Glycine max - genetics ; Physiological aspects ; Plant Proteins - genetics ; Polymerase chain reaction ; Reference Standards ; Reverse Transcriptase Polymerase Chain Reaction - methods ; Reverse Transcriptase Polymerase Chain Reaction - standards ; Software packages ; Soybean ; Statistical analysis</subject><ispartof>BMC molecular biology, 2009-09, Vol.10 (1), p.93-93, Article 93</ispartof><rights>COPYRIGHT 2009 BioMed Central Ltd.</rights><rights>2009 Hu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</rights><rights>Copyright © 2009 Hu et al; licensee BioMed Central Ltd. 2009 Hu et al; licensee BioMed Central Ltd.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-b651t-e574cdf72bf172c4190bcb248ec1697a79087294343a370e6ed8d426f93a6e963</citedby><cites>FETCH-LOGICAL-b651t-e574cdf72bf172c4190bcb248ec1697a79087294343a370e6ed8d426f93a6e963</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/PMC2761916/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2761916/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,881,24780,27901,27902,53766,53768,75707,75708</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/19785741$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Hu, Ruibo</creatorcontrib><creatorcontrib>Fan, Chengming</creatorcontrib><creatorcontrib>Li, Hongyu</creatorcontrib><creatorcontrib>Zhang, Qingzhu</creatorcontrib><creatorcontrib>Fu, Yong-Fu</creatorcontrib><title>Evaluation of putative reference genes for gene expression normalization in soybean by quantitative real-time RT-PCR</title><title>BMC molecular biology</title><addtitle>BMC Mol Biol</addtitle><description>Real-time quantitative reverse transcription PCR (RT-qPCR) data needs to be normalized for its proper interpretation. Housekeeping genes are routinely employed for this purpose, but their expression level cannot be assumed to remain constant under all possible experimental conditions. Thus, a systematic validation of reference genes is required to ensure proper normalization. For soybean, only a small number of validated reference genes are available to date.
A systematic comparison of 14 potential reference genes for soybean is presented. These included seven commonly used (ACT2, ACT11, TUB4, TUA5, CYP, UBQ10, EF1b) and seven new candidates (SKIP16, MTP, PEPKR1, HDC, TIP41, UKN1, UKN2). Expression stability was examined by RT-qPCR across 116 biological samples, representing tissues at various developmental stages, varied photoperiodic treatments, and a range of soybean cultivars. Expression of all 14 genes was variable to some extent, but that of SKIP16, UKN1 and UKN2 was overall the most stable. A combination of ACT11, UKN1 and UKN2 would be appropriate as a reference panel for normalizing gene expression data among different tissues, whereas the combination SKIP16, UKN1 and MTP was most suitable for developmental stages. ACT11, TUA5 and TIP41 were the most stably expressed when the photoperiod was altered, and TIP41, UKN1 and UKN2 when the light quality was changed. For six different cultivars in long day (LD) and short day (SD), their expression stability did not vary significantly with ACT11, UKN2 and TUB4 being the most stable genes. The relative gene expression level of GmFTL3, an ortholog of Arabidopsis FT (FLOWERING LOCUS T) was detected to validate the reference genes selected in this study.
None of the candidate reference genes was uniformly expressed across all experimental conditions, and the most suitable reference genes are conditional-, tissue-specific-, developmental-, and cultivar-dependent. Most of the new reference genes performed better than the conventional housekeeping genes. These results should guide the selection of reference genes for gene expression studies in soybean.</description><subject>Analysis</subject><subject>Arabidopsis</subject><subject>Crop science</subject><subject>Cytochrome P-450</subject><subject>Data processing</subject><subject>Gene expression</subject><subject>Gene Expression Profiling</subject><subject>Gene Expression Regulation, Plant</subject><subject>Genes, Plant - genetics</subject><subject>Genetic aspects</subject><subject>Genomes</subject><subject>Glycine max - genetics</subject><subject>Physiological aspects</subject><subject>Plant Proteins - genetics</subject><subject>Polymerase chain reaction</subject><subject>Reference Standards</subject><subject>Reverse Transcriptase Polymerase Chain Reaction - methods</subject><subject>Reverse Transcriptase Polymerase Chain Reaction - standards</subject><subject>Software packages</subject><subject>Soybean</subject><subject>Statistical analysis</subject><issn>1471-2199</issn><issn>1471-2199</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2009</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><recordid>eNp9kk1v1DAQhiMEoqVw5oYicQAOaW3H649LpbIqUKkSaClny_FOFleJvbWdVZdfj0NWSxcB8sGjmWdejV9PUbzE6BRjwc4w5bgiWMoKo0rWj4rjfebxg_ioeBbjLUKYi1o8LY6w5GLGKT4u0uVGd4NO1rvSt-V6SDneQBmghQDOQLkCB7FsffgVlXC_DhDjyDsfet3ZH1O3dWX02wa0K5tteTdol-xeTHdVsj2Ui5vqy3zxvHjS6i7Ci919Unz7cHkz_1Rdf_54Nb-4rho2w6mCPKJZtpw0LebEUCxRYxpCBRjMJNdcIsGJpDWtdc0RMFiKJSWslbVmIFl9UpxPuuuh6WFpwKWgO7UOttdhq7y26rDi7He18htFOMMSjwLvJ4HG-n8IHFaM79XouhpdVxgpWWeRN7spgr8bICbV22ig67QDP0TFa4oEI5hn8u1_SUwlYVQKjjP6-g_01g_BZTczhdAMcSZEpk4naqU7UNa1Pk9p8llCb4130NqcvyBIMkopI7nh3UFDZhLcp5UeYlRXXxeH7NnEmuBjzAuz9yW_e9zOvzjx6uF__OZ361j_BCT34CY</recordid><startdate>20090928</startdate><enddate>20090928</enddate><creator>Hu, Ruibo</creator><creator>Fan, Chengming</creator><creator>Li, Hongyu</creator><creator>Zhang, Qingzhu</creator><creator>Fu, Yong-Fu</creator><general>BioMed Central Ltd</general><general>BioMed Central</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>ISR</scope><scope>3V.</scope><scope>7TM</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M7P</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20090928</creationdate><title>Evaluation of putative reference genes for gene expression normalization in soybean by quantitative real-time RT-PCR</title><author>Hu, Ruibo ; 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Housekeeping genes are routinely employed for this purpose, but their expression level cannot be assumed to remain constant under all possible experimental conditions. Thus, a systematic validation of reference genes is required to ensure proper normalization. For soybean, only a small number of validated reference genes are available to date.
A systematic comparison of 14 potential reference genes for soybean is presented. These included seven commonly used (ACT2, ACT11, TUB4, TUA5, CYP, UBQ10, EF1b) and seven new candidates (SKIP16, MTP, PEPKR1, HDC, TIP41, UKN1, UKN2). Expression stability was examined by RT-qPCR across 116 biological samples, representing tissues at various developmental stages, varied photoperiodic treatments, and a range of soybean cultivars. Expression of all 14 genes was variable to some extent, but that of SKIP16, UKN1 and UKN2 was overall the most stable. A combination of ACT11, UKN1 and UKN2 would be appropriate as a reference panel for normalizing gene expression data among different tissues, whereas the combination SKIP16, UKN1 and MTP was most suitable for developmental stages. ACT11, TUA5 and TIP41 were the most stably expressed when the photoperiod was altered, and TIP41, UKN1 and UKN2 when the light quality was changed. For six different cultivars in long day (LD) and short day (SD), their expression stability did not vary significantly with ACT11, UKN2 and TUB4 being the most stable genes. The relative gene expression level of GmFTL3, an ortholog of Arabidopsis FT (FLOWERING LOCUS T) was detected to validate the reference genes selected in this study.
None of the candidate reference genes was uniformly expressed across all experimental conditions, and the most suitable reference genes are conditional-, tissue-specific-, developmental-, and cultivar-dependent. Most of the new reference genes performed better than the conventional housekeeping genes. These results should guide the selection of reference genes for gene expression studies in soybean.</abstract><cop>England</cop><pub>BioMed Central Ltd</pub><pmid>19785741</pmid><doi>10.1186/1471-2199-10-93</doi><tpages>1</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Analysis Arabidopsis Crop science Cytochrome P-450 Data processing Gene expression Gene Expression Profiling Gene Expression Regulation, Plant Genes, Plant - genetics Genetic aspects Genomes Glycine max - genetics Physiological aspects Plant Proteins - genetics Polymerase chain reaction Reference Standards Reverse Transcriptase Polymerase Chain Reaction - methods Reverse Transcriptase Polymerase Chain Reaction - standards Software packages Soybean Statistical analysis |
| title | Evaluation of putative reference genes for gene expression normalization in soybean by quantitative real-time RT-PCR |
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