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About stable combinations of non-stable genes as reference genes for RT-qPCR data normalization
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International audience. Gene expression profiling is of key importance in all domains of life sciences, as medicine, environment, and plants, for both basic and applied research. In spite of the emergence of microarrays and high-throughput sequencing, qPCR still remains a standard method for gene expression analyses. For this purpose, qPCR data normalization is a crucial step. Among normalization methods, the use of reference genes, supposed to be stable all along experimental conditions, is required. In the present study, we show that a stable combination of non-stable genes outperforms standard reference genes for qPCR data normalization. A stable combination of genes consists on a fixed number of genes which expressions balance each other all along experimental conditions of interest. Moreover, the present study shows that such an optimal combination of genes can be found using a comprehensive database of RNA-Seq data. As a case study, this new method has been developed using the tomato model plant, with corresponding RNA-Seq data from the TomExpress database. Our results demonstrate the superiority of our method over commonly used housekeeping genes or other stably-expressed genes. We therefore recommend the use of our new method together with classic ones, in order to always obtain best reference genes for a given experimental design.
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