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Background  Quantitative RT-PCR is the method of choice for studying, with both sensitivity and accuracy, the expression of genes. A reliable normalization of the data, using several reference genes, is critical for an accurate quantification of gene expression. Here, we propose a set of reference genes, of the phytopathogenic bacteria  Dickeya dadantii  and  Pectobacterium atrosepticum , which are stable in a wide range of growth conditions.    Results  We extracted, from a  D. dadantii  micro-array transcript profile dataset comprising thirty-two different growth conditions, an initial set of 49 expressed genes with very low variation in gene expression. Out of these, we retained 10 genes representing different functional categories, different levels of expression (low, medium, and high) and with no systematic variation in expression correlating with growth conditions. We measured the expression of these reference gene candidates using quantitative RT-PCR in 50 different experimental conditions, mimicking the environment encountered by the bacteria in their host and directly during the infection process  in planta . The two most stable genes (ABF-0017965 ( lpxC ) and ABF-0020529 ( yafS ) were successfully used for normalization of RT-qPCR data. Finally, we demonstrated that the ortholog of  lpxC  and  yafS  in  Pectobacterium atrosepticum  also showed stable expression in diverse growth conditions.    Conclusions  We have identified at least two genes,  lpxC  (ABF-0017965) and  yafS  (ABF-0020509), whose expressions are stable in a wide range of growth conditions and during infection. Thus, these genes are considered suitable for use as reference genes for the normalization of real-time RT-qPCR data of the two main pectinolytic phytopathogenic bacteria  D. dadantii  and  P. atrosepticum  and, probably, of other  Enterobacteriaceae . Moreover, we defined general criteria to select good reference genes in bacteria.

lpxC and yafS are the Most Suitable Internal Controls to Normalize Real Time RT-qPCR Expression in the Phytopathogenic Bacteria Dickeya dadantii