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Abstract
Quantitative real-time RT-PCR (qRT-PCR) has become one of the most widely used methods for accurate quantification of gene expression. Since there are no universal reference genes for normalization, the optimal strategy to normalize raw qRT-PCR data is to perform an initial comparison of a set of independent reference genes to assess the most stable ones in each biological model. Normalization of a qRT-PCR experiment helps to ensure that the results are both statistically significant and biologically meaningful. Tomato is the model of choice to study fleshy fruit development. The miniature tomato (Solanum lycopersicum L.) cultivar Micro-Tom (MT) is considered a model system for tomato genetics and functional genomics. A new genotype, containing the Rg1 allele, improves tomato in vitro regeneration. In this work, we evaluated the expression stability of four tomato reference genes, namely CAC, SAND, Expressed, and ACTIN2. We showed that the genes CAC and Exp are the best reference genes of the four we tested during fruit development in the MT-Rg1 genotype. Furthermore, we validated the reference genes by showing that the expression profiles of the transcription factors FRUITFULL1 and APETALA2c during fruit development are comparable to previous reports using other tomato cultivars.
Highlights
Reverse transcription followed by quantitative PCR assay is an extremely sensitive technique that provides accurate and reproducible quantification of nucleic acids based on the exponential incorporation of fluorescent molecules into genetic material (Gachon et al, 2004; Nolan et al, 2006)
Based on previous studies conducted on S. lycopersicum cv. ciliegia (Expósito-Rodríguez et al, 2008), a total of four candidate reference genes were selected for Quantitative real-time RT-PCR (qRT-PCR) normalization
Several reports have shown the importance of selecting proper reference genes for data normalization, and how the identity of these genes will vary depending on the model of study (Jain et al, 2006; Expósito-Rodríguez et al, 2008; Jian et al, 2008; Manoli et al, 2012; Lambret-Frotté et al, 2015; Kanakachari et al, 2016)
Summary
Reverse transcription followed by quantitative PCR (qRT-PCR) assay is an extremely sensitive technique that provides accurate and reproducible quantification of nucleic acids based on the exponential incorporation of fluorescent molecules into genetic material (Gachon et al, 2004; Nolan et al, 2006). QRT-PCR analysis has become the method of choice for gene expression studies and validating transcriptomic data. One of the most crucial points in RT-qPCR data analysis is the choice of a proper normalization method. The parallel quantification of endogenous reference genes is accepted as Selection of Reference Genes in Tomato Fruit the most reliable method for sample normalization. The normalization of relative quantities with reference genes relies on the assumption that the reference genes are stably expressed across all tested samples, and that they are not being significantly altered across treatments or conditions (Vandesompele et al, 2002). Several reports have shown that the transcript levels of commonly used reference genes, known as housekeeping genes, can vary considerably under different experimental conditions (e.g., Thellin et al, 1999; Suzuki et al, 2000; Czechowski et al, 2005; Jain et al, 2006). A reference gene with stable expression in one organism may not be suitable for normalization of gene expression in another organism (e.g., Jain et al, 2006; Jian et al, 2008; Manoli et al, 2012; LambretFrotté et al, 2015; Kanakachari et al, 2016)
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