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首页> 外文期刊>Protoplasma: An International Journal of Cell Biology >A spiking strategy facilitates housekeeping selection for RT-qPCR analysis under different biotic stresses in eggplant
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A spiking strategy facilitates housekeeping selection for RT-qPCR analysis under different biotic stresses in eggplant

机译：尖峰策略促进了在茄子中不同生物应力下的RT-QPCR分析的家务选择

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摘要

Endogenous housekeeping genes are traditionally employed to normalize the expression of target genes in RT-qPCR studies. Assuming that a perfect housekeeping suitable for every condition does not exist, expression stability of the chosen reference gene should be evaluated at every new experiment. The housekeeping selection process reveals furthermore complicated and time-consuming when different conditions have to be compared in the same experimental dataset. As an alternative strategy, we spiked an external reference transcript (ERT) into all RNA samples of our dataset (eggplant roots subjected to different biotic stresses), and used it to normalize the expression levels of native candidate housekeeping. ERT expression resulted highly stable across all samples and enabled to indicate glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the most stable endogenous housekeeping. This result was confirmed by the use of GeNorm, Normfinder, and BestKeeper algorithms. This method might be generally applied to expedite the selection process of the best reference gene.
机译：传统上使用内源性家务基因来规范靶基因在RT-QPCR研究中的表达。假设不存在适合所有条件的完美内务监狱，应在每次新实验中评估所选参考基因的表达稳定性。家务选择过程还揭示了当必须在同一实验数据集中比较不同的条件时复杂且耗时。作为替代策略，我们将外部参考转录物（ERT）掺入了我们数据集的所有RNA样本（经过不同的生物应力的茄子根），并用它来规范化本地候选人的表达水平。 ERT表达在所有样品中产生高度稳定性，并且能够表明甘油醛3-磷酸脱氢酶（GAPDH）作为最稳定的内源内良好管家。通过使用Genorm，Normfinder和BestKeeper算法确认了该结果。该方法通常可以应用于加快最佳参考基因的选择过程。

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