Selection and evaluation of reference genes for qRT-PCR analysis in Euscaphis konishii Hayata based on transcriptome data
Abstract Background Quantitative real-time reverse transcription-polymerase chain reaction has been widely used in gene expression analysis, however, to have reliable and accurate results, reference genes are necessary to normalize gene expression under different experimental conditions. Several rel...
| Main Authors: | , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2018-06-01
|
| Series: | Plant Methods |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s13007-018-0311-x |
| _version_ | 1819065578527129600 |
|---|---|
| author | Wenxian Liang Xiaoxing Zou Rebeca Carballar-Lejarazú Lingjiao Wu Weihong Sun Xueyuan Yuan Songqing Wu Pengfei Li Hui Ding Lin Ni Wei Huang Shuangquan Zou |
| author_facet | Wenxian Liang Xiaoxing Zou Rebeca Carballar-Lejarazú Lingjiao Wu Weihong Sun Xueyuan Yuan Songqing Wu Pengfei Li Hui Ding Lin Ni Wei Huang Shuangquan Zou |
| author_sort | Wenxian Liang |
| collection | DOAJ |
| description | Abstract Background Quantitative real-time reverse transcription-polymerase chain reaction has been widely used in gene expression analysis, however, to have reliable and accurate results, reference genes are necessary to normalize gene expression under different experimental conditions. Several reliable reference genes have been reported in plants of Traditional Chinese Medicine, but none have been identified for Euscaphis konishii Hayata. Results In this study, 12 candidate reference genes, including 3 common housekeeping genes and 9 novel genes based on E. konishii Hayata transcriptome data were selected and analyzed in different tissues (root, branch, leaf, capsule and seed), capsule and seed development stages. Expression stability was calculated using geNorm and NormFinder, the minimal number of reference genes required for accurate normalization was calculated by Vn/Vn + 1 using geNorm. EkEEF-5A-1 and EkADF2 were the two most stable reference genes for all samples, while EkGSTU1 and EkGAPDH were the most stable reference genes for tissue samples. For seed development stages, EkGAPDH and EkEEF-5A-1 were the most stable genes, whereas EkGSTU1 and EkGAPDH were identified as the two most stable genes in the capsule development stages. Two reference genes were sufficient to normalize gene expression across all sample sets. Conclusion Results of this study revealed that suitable reference genes should be selected for different experimental samples, and not all the common reference genes are suitable for different tissue samples and/or experimental conditions. In this study, we present the first data of reference genes selection for E. konishii Hayata based on transcriptome data, our data will facilitate further studies in molecular biology and gene function on E. konishii Hayata and other closely related species. |
| first_indexed | 2024-12-21T15:48:42Z |
| format | Article |
| id | doaj.art-6472cdec57b943249b0c21760c48f518 |
| institution | Directory Open Access Journal |
| issn | 1746-4811 |
| language | English |
| last_indexed | 2024-12-21T15:48:42Z |
| publishDate | 2018-06-01 |
| publisher | BMC |
| record_format | Article |
| series | Plant Methods |
| spelling | doaj.art-6472cdec57b943249b0c21760c48f5182022-12-21T18:58:17ZengBMCPlant Methods1746-48112018-06-011411910.1186/s13007-018-0311-xSelection and evaluation of reference genes for qRT-PCR analysis in Euscaphis konishii Hayata based on transcriptome dataWenxian Liang0Xiaoxing Zou1Rebeca Carballar-Lejarazú2Lingjiao Wu3Weihong Sun4Xueyuan Yuan5Songqing Wu6Pengfei Li7Hui Ding8Lin Ni9Wei Huang10Shuangquan Zou11College of Forestry, Fujian Agriculture and Forestry UniversityCollege of Forestry, Fujian Agriculture and Forestry UniversityDepartment of Microbiology and Molecular Genetics, University of CaliforniaCollege of Forestry, Fujian Agriculture and Forestry UniversityCollege of Forestry, Fujian Agriculture and Forestry UniversityCollege of Forestry, Fujian Agriculture and Forestry UniversityCollege of Forestry, Fujian Agriculture and Forestry UniversityCollege of Forestry, Fujian Agriculture and Forestry UniversityCollege of Forestry, Fujian Agriculture and Forestry UniversityFujian Colleges and Universities Engineering Research Institute of Conservation and Utilization of Natural Bioresources, Fujian Agriculture and Forestry UniversityFujian Colleges and Universities Engineering Research Institute of Conservation and Utilization of Natural Bioresources, Fujian Agriculture and Forestry UniversityCollege of Forestry, Fujian Agriculture and Forestry UniversityAbstract Background Quantitative real-time reverse transcription-polymerase chain reaction has been widely used in gene expression analysis, however, to have reliable and accurate results, reference genes are necessary to normalize gene expression under different experimental conditions. Several reliable reference genes have been reported in plants of Traditional Chinese Medicine, but none have been identified for Euscaphis konishii Hayata. Results In this study, 12 candidate reference genes, including 3 common housekeeping genes and 9 novel genes based on E. konishii Hayata transcriptome data were selected and analyzed in different tissues (root, branch, leaf, capsule and seed), capsule and seed development stages. Expression stability was calculated using geNorm and NormFinder, the minimal number of reference genes required for accurate normalization was calculated by Vn/Vn + 1 using geNorm. EkEEF-5A-1 and EkADF2 were the two most stable reference genes for all samples, while EkGSTU1 and EkGAPDH were the most stable reference genes for tissue samples. For seed development stages, EkGAPDH and EkEEF-5A-1 were the most stable genes, whereas EkGSTU1 and EkGAPDH were identified as the two most stable genes in the capsule development stages. Two reference genes were sufficient to normalize gene expression across all sample sets. Conclusion Results of this study revealed that suitable reference genes should be selected for different experimental samples, and not all the common reference genes are suitable for different tissue samples and/or experimental conditions. In this study, we present the first data of reference genes selection for E. konishii Hayata based on transcriptome data, our data will facilitate further studies in molecular biology and gene function on E. konishii Hayata and other closely related species.http://link.springer.com/article/10.1186/s13007-018-0311-xEuscaphis konishii HayataReference geneqRT-PCRTranscriptomeGene expressionNormalization |
| spellingShingle | Wenxian Liang Xiaoxing Zou Rebeca Carballar-Lejarazú Lingjiao Wu Weihong Sun Xueyuan Yuan Songqing Wu Pengfei Li Hui Ding Lin Ni Wei Huang Shuangquan Zou Selection and evaluation of reference genes for qRT-PCR analysis in Euscaphis konishii Hayata based on transcriptome data Plant Methods Euscaphis konishii Hayata Reference gene qRT-PCR Transcriptome Gene expression Normalization |
| title | Selection and evaluation of reference genes for qRT-PCR analysis in Euscaphis konishii Hayata based on transcriptome data |
| title_full | Selection and evaluation of reference genes for qRT-PCR analysis in Euscaphis konishii Hayata based on transcriptome data |
| title_fullStr | Selection and evaluation of reference genes for qRT-PCR analysis in Euscaphis konishii Hayata based on transcriptome data |
| title_full_unstemmed | Selection and evaluation of reference genes for qRT-PCR analysis in Euscaphis konishii Hayata based on transcriptome data |
| title_short | Selection and evaluation of reference genes for qRT-PCR analysis in Euscaphis konishii Hayata based on transcriptome data |
| title_sort | selection and evaluation of reference genes for qrt pcr analysis in euscaphis konishii hayata based on transcriptome data |
| topic | Euscaphis konishii Hayata Reference gene qRT-PCR Transcriptome Gene expression Normalization |
| url | http://link.springer.com/article/10.1186/s13007-018-0311-x |
| work_keys_str_mv | AT wenxianliang selectionandevaluationofreferencegenesforqrtpcranalysisineuscaphiskonishiihayatabasedontranscriptomedata AT xiaoxingzou selectionandevaluationofreferencegenesforqrtpcranalysisineuscaphiskonishiihayatabasedontranscriptomedata AT rebecacarballarlejarazu selectionandevaluationofreferencegenesforqrtpcranalysisineuscaphiskonishiihayatabasedontranscriptomedata AT lingjiaowu selectionandevaluationofreferencegenesforqrtpcranalysisineuscaphiskonishiihayatabasedontranscriptomedata AT weihongsun selectionandevaluationofreferencegenesforqrtpcranalysisineuscaphiskonishiihayatabasedontranscriptomedata AT xueyuanyuan selectionandevaluationofreferencegenesforqrtpcranalysisineuscaphiskonishiihayatabasedontranscriptomedata AT songqingwu selectionandevaluationofreferencegenesforqrtpcranalysisineuscaphiskonishiihayatabasedontranscriptomedata AT pengfeili selectionandevaluationofreferencegenesforqrtpcranalysisineuscaphiskonishiihayatabasedontranscriptomedata AT huiding selectionandevaluationofreferencegenesforqrtpcranalysisineuscaphiskonishiihayatabasedontranscriptomedata AT linni selectionandevaluationofreferencegenesforqrtpcranalysisineuscaphiskonishiihayatabasedontranscriptomedata AT weihuang selectionandevaluationofreferencegenesforqrtpcranalysisineuscaphiskonishiihayatabasedontranscriptomedata AT shuangquanzou selectionandevaluationofreferencegenesforqrtpcranalysisineuscaphiskonishiihayatabasedontranscriptomedata |