Expression Stabilities of Candidate Reference Genes for RT-qPCR in Chinese Jujube (Ziziphus jujuba Mill.) under a Variety of Conditions
Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) is a powerful method for evaluating patterns of gene expression. Jujube whole-genome sequencing has been completed, and analysis of gene function, an important part of any follow-up study, requires the appropriate selec...
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Published in | PloS one Vol. 11; no. 4; p. e0154212 |
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26.04.2016
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Abstract | Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) is a powerful method for evaluating patterns of gene expression. Jujube whole-genome sequencing has been completed, and analysis of gene function, an important part of any follow-up study, requires the appropriate selection of reference genes. Indeed, suitable reference gene selection for RT-qPCR is critical for accurate normalization of target gene expression. In this study, the software packages geNorm and NormFinder were employed to examine the expression stabilities of nine candidate reference genes under a variety of conditions. Actin-depolymerizing factor 1 (ACT1), Histone-H3 (His3), and Polyadenylate-binding protein-interacting protein (PAIP) were determined to be the most stably expressed genes during five stages of fruit development and ACT1, SiR-Fd, BTF3, and Tubulin alpha chain (TUA) across different tissues/organs. Whereas ACT1, Basic Transcription factor 3 (BTF3), Glyceraldehyde-3-phosphate dehydrogenase (GADPH), and PAIP were the most stable under dark conditions. ACT1, PAIP, BTF3, and Elongation factor 1- gamma (EF1γ) were the most stably expressed genes under phytoplasma infection. Among these genes, SiR-Fd and PAIP are here first reported as stable reference genes. When normalized using these most stable reference genes, the expression patterns of four target genes were found to be in accordance with physiological data, indicating that the reference genes selected in our study are suitable for use in such analyses. This study provides appropriate reference genes and corresponding primers for further RT-qPCR studies in Chinese jujube and emphasizes the importance of validating reference genes for gene expression analysis under variable experimental conditions. |
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AbstractList | Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) is a powerful method for evaluating patterns of gene expression. Jujube whole-genome sequencing has been completed, and analysis of gene function, an important part of any follow-up study, requires the appropriate selection of reference genes. Indeed, suitable reference gene selection for RT-qPCR is critical for accurate normalization of target gene expression. In this study, the software packages geNorm and NormFinder were employed to examine the expression stabilities of nine candidate reference genes under a variety of conditions. Actin-depolymerizing factor 1 (ACT1), Histone-H3 (His3), and Polyadenylate-binding protein-interacting protein (PAIP) were determined to be the most stably expressed genes during five stages of fruit development and ACT1, SiR-Fd, BTF3, and Tubulin alpha chain (TUA) across different tissues/organs. Whereas ACT1, Basic Transcription factor 3 (BTF3), Glyceraldehyde-3-phosphate dehydrogenase (GADPH), and PAIP were the most stable under dark conditions. ACT1, PAIP, BTF3, and Elongation factor 1- gamma (EF1γ) were the most stably expressed genes under phytoplasma infection. Among these genes, SiR-Fd and PAIP are here first reported as stable reference genes. When normalized using these most stable reference genes, the expression patterns of four target genes were found to be in accordance with physiological data, indicating that the reference genes selected in our study are suitable for use in such analyses. This study provides appropriate reference genes and corresponding primers for further RT-qPCR studies in Chinese jujube and emphasizes the importance of validating reference genes for gene expression analysis under variable experimental conditions. Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) is a powerful method for evaluating patterns of gene expression. Jujube whole-genome sequencing has been completed, and analysis of gene function, an important part of any follow-up study, requires the appropriate selection of reference genes. Indeed, suitable reference gene selection for RT-qPCR is critical for accurate normalization of target gene expression. In this study, the software packages geNorm and NormFinder were employed to examine the expression stabilities of nine candidate reference genes under a variety of conditions. Actin-depolymerizing factor 1 (ACT1), Histone-H3 (His3), and Polyadenylate-binding protein-interacting protein (PAIP) were determined to be the most stably expressed genes during five stages of fruit development and ACT1, SiR-Fd, BTF3, and Tubulin alpha chain (TUA) across different tissues/organs. Whereas ACT1, Basic Transcription factor 3 (BTF3), Glyceraldehyde-3-phosphate dehydrogenase (GADPH), and PAIP were the most stable under dark conditions. ACT1, PAIP, BTF3, and Elongation factor 1- gamma (EF1[gamma]) were the most stably expressed genes under phytoplasma infection. Among these genes, SiR-Fd and PAIP are here first reported as stable reference genes. When normalized using these most stable reference genes, the expression patterns of four target genes were found to be in accordance with physiological data, indicating that the reference genes selected in our study are suitable for use in such analyses. This study provides appropriate reference genes and corresponding primers for further RT-qPCR studies in Chinese jujube and emphasizes the importance of validating reference genes for gene expression analysis under variable experimental conditions. Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) is a powerful method for evaluating patterns of gene expression. Jujube whole-genome sequencing has been completed, and analysis of gene function, an important part of any follow-up study, requires the appropriate selection of reference genes. Indeed, suitable reference gene selection for RT-qPCR is critical for accurate normalization of target gene expression. In this study, the software packages geNorm and NormFinder were employed to examine the expression stabilities of nine candidate reference genes under a variety of conditions. Actin-depolymerizing factor 1 ( ACT1 ), Histone-H3 ( His3 ), and Polyadenylate-binding protein-interacting protein ( PAIP ) were determined to be the most stably expressed genes during five stages of fruit development and ACT1 , SiR-Fd , BTF3 , and Tubulin alpha chain ( TUA ) across different tissues/organs. Whereas ACT1 , Basic Transcription factor 3 ( BTF3 ), Glyceraldehyde-3-phosphate dehydrogenase ( GADPH ), and PAIP were the most stable under dark conditions. ACT1 , PAIP , BTF3 , and Elongation factor 1- gamma ( EF1γ ) were the most stably expressed genes under phytoplasma infection. Among these genes, SiR-Fd and PAIP are here first reported as stable reference genes. When normalized using these most stable reference genes, the expression patterns of four target genes were found to be in accordance with physiological data, indicating that the reference genes selected in our study are suitable for use in such analyses. This study provides appropriate reference genes and corresponding primers for further RT-qPCR studies in Chinese jujube and emphasizes the importance of validating reference genes for gene expression analysis under variable experimental conditions. |
Audience | Academic |
Author | Bu, Jiaodi Zhao, Jin Liu, Mengjun |
AuthorAffiliation | 1 College of Life Science, Agricultural University of Hebei, Baoding, Hebei, China 2 Research Center of Chinese Jujube, Agricultural University of Hebei, Baoding, Hebei, China Beijing Forestry University, CHINA |
AuthorAffiliation_xml | – name: Beijing Forestry University, CHINA – name: 2 Research Center of Chinese Jujube, Agricultural University of Hebei, Baoding, Hebei, China – name: 1 College of Life Science, Agricultural University of Hebei, Baoding, Hebei, China |
Author_xml | – sequence: 1 givenname: Jiaodi surname: Bu fullname: Bu, Jiaodi organization: College of Life Science, Agricultural University of Hebei, Baoding, Hebei, China – sequence: 2 givenname: Jin surname: Zhao fullname: Zhao, Jin organization: College of Life Science, Agricultural University of Hebei, Baoding, Hebei, China – sequence: 3 givenname: Mengjun surname: Liu fullname: Liu, Mengjun organization: Research Center of Chinese Jujube, Agricultural University of Hebei, Baoding, Hebei, China |
BackLink | https://www.ncbi.nlm.nih.gov/pubmed/27116123$$D View this record in MEDLINE/PubMed |
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Notes | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: JZ. Performed the experiments: JZ JB. Analyzed the data: JZ JB. Contributed reagents/materials/analysis tools: JZ ML. Wrote the paper: JZ JB. |
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SubjectTerms | Actin Actin-depolymerizing protein Biology and Life Sciences Cyanobacteria Depolymerization Developmental stages Elongation Fruits Gene Expression Gene Expression Profiling - methods Gene Expression Regulation, Plant Gene sequencing Genes Genes, Plant Genetic aspects Genomes Glyceraldehyde-3-phosphate dehydrogenase Jujube Life sciences Organs Phosphates Physiological aspects Phytoplasma Plant Proteins - genetics Plant sciences Polyadenylation Polymerase chain reaction Primers Research and Analysis Methods Reverse Transcriptase Polymerase Chain Reaction - methods Reverse transcription Software packages Studies Tissues Tubulin Ziziphus Ziziphus - genetics Ziziphus jujuba |
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Title | Expression Stabilities of Candidate Reference Genes for RT-qPCR in Chinese Jujube (Ziziphus jujuba Mill.) under a Variety of Conditions |
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