Barbierato Valeria, Sala Tea, Rinaldi Paola, Bassolino Laura, Barchi Lorenzo, Rotino Giuseppe Leonardo, Toppino Laura
CREA- Consiglio per la ricerca in agricoltura e l'analisi dell'economia agraria, Unità di Ricerca per l'Orticoltura, Via Paullese, 28 Montanaso Lombardo, 26836, Lodi, Italy.
DISAFA Plant Genetics and Breeding, University of Torino, Grugliasco, 10095, Torino, Italy.
Protoplasma. 2017 Nov;254(6):2215-2223. doi: 10.1007/s00709-017-1111-2. Epub 2017 Apr 21.
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.
在逆转录定量聚合酶链反应（RT-qPCR）研究中，传统上使用内源性管家基因来标准化目标基因的表达。假设不存在适用于每种情况的完美管家基因，那么在每个新实验中都应评估所选参考基因的表达稳定性。当必须在同一实验数据集中比较不同条件时，管家基因的选择过程会变得更加复杂和耗时。作为一种替代策略，我们将外部参考转录本（ERT）掺入我们数据集的所有RNA样本（遭受不同生物胁迫的茄子根）中，并使用它来标准化天然候选管家基因的表达水平。ERT表达在所有样本中高度稳定，并能够将甘油醛-3-磷酸脱氢酶（GAPDH）指示为最稳定的内源性管家基因。使用GeNorm、Normfinder和BestKeeper算法证实了这一结果。该方法可能普遍适用于加快最佳参考基因的选择过程。
