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Selection and evaluation of reference genes for qRT-PCR analysis in Euscaphis konishii Hayata based on transcriptome data.
Plant Methods ( IF 4.7 ) Pub Date : 2018-06-09 , DOI: 10.1186/s13007-018-0311-x Wenxian Liang 1, 2 , Xiaoxing Zou 1, 2 , Rebeca Carballar-Lejarazú 3 , Lingjiao Wu 1, 2 , Weihong Sun 1, 2 , Xueyuan Yuan 1, 2 , Songqing Wu 1, 2 , Pengfei Li 1, 2 , Hui Ding 1, 2 , Lin Ni 2, 4 , Wei Huang 2, 5 , Shuangquan Zou 1, 2
Plant Methods ( IF 4.7 ) Pub Date : 2018-06-09 , DOI: 10.1186/s13007-018-0311-x Wenxian Liang 1, 2 , Xiaoxing Zou 1, 2 , Rebeca Carballar-Lejarazú 3 , Lingjiao Wu 1, 2 , Weihong Sun 1, 2 , Xueyuan Yuan 1, 2 , Songqing Wu 1, 2 , Pengfei Li 1, 2 , Hui Ding 1, 2 , Lin Ni 2, 4 , Wei Huang 2, 5 , Shuangquan Zou 1, 2
Affiliation
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.
中文翻译：
基于转录组数据选择和评估用于 qRT-PCR 分析的 Euscaphis konishii Hayata 的参考基因。
背景定量实时逆转录聚合酶链反应已广泛用于基因表达分析，然而，为了获得可靠和准确的结果，需要参考基因来标准化不同实验条件下的基因表达。在中药植物中已经报道了几个可靠的参考基因，但尚未鉴定出Euscaphis konishii Hayata。结果在本研究中，基于E. konishii Hayata转录组数据，筛选出12个候选参考基因，包括3个常见管家基因和9个新基因，并在不同组织（根、枝、叶、荚膜和种子）、荚膜和种子发育中进行分析。阶段。使用 geNorm 和 NormFinder 计算表达稳定性，使用 geNorm 通过 Vn/Vn + 1 计算准确标准化所需的最小参考基因数量。EkEEF-5A-1 和 EkADF2 是所有样品中最稳定的两个参考基因，而 EkGSTU1 和 EkGAPDH 是组织样品中最稳定的参考基因。对于种子发育阶段，EkGAPDH 和 EkEEF-5A-1 是最稳定的基因，而 EkGSTU1 和 EkGAPDH 被确定为蒴果发育阶段最稳定的两个基因。两个参考基因足以使所有样本集的基因表达正常化。结论本研究结果表明，应为不同的实验样本选择合适的参考基因，并非所有常见的参考基因都适用于不同的组织样本和/或实验条件。在这项研究中，
更新日期：2018-06-04
中文翻译：
基于转录组数据选择和评估用于 qRT-PCR 分析的 Euscaphis konishii Hayata 的参考基因。
背景定量实时逆转录聚合酶链反应已广泛用于基因表达分析，然而，为了获得可靠和准确的结果，需要参考基因来标准化不同实验条件下的基因表达。在中药植物中已经报道了几个可靠的参考基因，但尚未鉴定出Euscaphis konishii Hayata。结果在本研究中，基于E. konishii Hayata转录组数据，筛选出12个候选参考基因，包括3个常见管家基因和9个新基因，并在不同组织（根、枝、叶、荚膜和种子）、荚膜和种子发育中进行分析。阶段。使用 geNorm 和 NormFinder 计算表达稳定性，使用 geNorm 通过 Vn/Vn + 1 计算准确标准化所需的最小参考基因数量。EkEEF-5A-1 和 EkADF2 是所有样品中最稳定的两个参考基因，而 EkGSTU1 和 EkGAPDH 是组织样品中最稳定的参考基因。对于种子发育阶段，EkGAPDH 和 EkEEF-5A-1 是最稳定的基因，而 EkGSTU1 和 EkGAPDH 被确定为蒴果发育阶段最稳定的两个基因。两个参考基因足以使所有样本集的基因表达正常化。结论本研究结果表明，应为不同的实验样本选择合适的参考基因，并非所有常见的参考基因都适用于不同的组织样本和/或实验条件。在这项研究中，
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