Abstract
BackgroundData normalization is a key step in gene expression analysis by qPCR. Endogenous control genes are used to estimate variations and experimental errors occurring during sample preparation and expression measurements. However, the transcription level of the most commonly used reference genes can vary considerably in samples obtained from different individuals, tissues, developmental stages and under variable physiological conditions, resulting in a misinterpretation of the performance of the target gene(s). This issue has been scarcely approached in woody species such as grapevine.ResultsA statistical criterion was applied to select a sub-set of 19 candidate reference genes from a total of 242 non-differentially expressed (NDE) genes derived from a RNA-Seq experiment comprising ca. 500 million reads obtained from 14 table-grape genotypes sampled at four phenological stages. From the 19 candidate reference genes, VvAIG1 (AvrRpt2-induced gene) and VvTCPB (T-complex 1 beta-like protein) were found to be the most stable ones after comparing the complete set of genotypes and phenological stages studied. This result was further validated by qPCR and geNorm analyses.ConclusionsBased on the evidence presented in this work, we propose to use the grapevine genes VvAIG1 or VvTCPB or both as a reference tool to normalize RNA expression in qPCR assays or other quantitative method intended to measure gene expression in berries and other tissues of this fruit crop, sampled at different developmental stages and physiological conditions.
Highlights
Data normalization is a key step in gene expression analysis by Quantitative real-time PCR (qPCR)
Identification of putative reference genes Usually the search for reference genes in any plant species is based on the identification of orthologs of genes stably expressed in model plants, mainly from Arabidopsis thaliana [44,45]
Even when the main outcome of an RNA-Seq exercise is the identification of differentially expressed genes, in this case the same data set was used to search for putative reference genes, considered as that any gene that has a minimal expression level variation in every sample analyzed
Summary
Data normalization is a key step in gene expression analysis by qPCR. Endogenous control genes are used to estimate variations and experimental errors occurring during sample preparation and expression measurements. The transcription level of the most commonly used reference genes can vary considerably in samples obtained from different individuals, tissues, developmental stages and under variable physiological conditions, resulting in a misinterpretation of the performance of the target gene(s). This issue has been scarcely approached in woody species such as grapevine. Several reports have demonstrated that transcript levels of these genes vary considerably under different experimental conditions and their suitability for gene expression studies must be evaluated case by case [22,33,34]. This implies that a reference gene with stable expression in one organism may not be suitable for normalization of gene expression in another organism [35,36], or even in different experiments for the same species
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