ИСТИНА

Real-time RT-PCR is a method for the analysis of gene expression widely used in different fields of biology. This approach can provide accurate quantitative data but has several issues that can greatly influence the results. Among them the most important is the normalization. The commonly used method of normalization is the use of reference gene – a gene whose expression is stable in control and experimental conditions. The comparison of gene expression level between different species is the integral part of many evo-devo studies. However no attempt was made to assess which reference gene (or genes) is better suited for this purpose. To address this question we took as a model five plant species from Brassicaceae, the family to which model plant species Arabidopsis thaliana belongs. A genome-wide survey of gene expression stability in A. thaliana revealed a lot of genes stably expressed during development and under different stress conditions. We have chosen four orthologs of the A. thaliana genes identified as the most stable in this genome-wide survey as candidate reference genes for interspecific comparison. Using these genes as reference we analyzed the expression of five genes involved in flower development and four genes involved in cold stress response. Our results indicate that significant difference in gene expression level is observed even in species with similar morphology. This should be taken into account when using quantitative real-time RT-PCR data for interspecific comparisons.