%0 Journal Article
%T Evaluation of reference genes for real-time RT-PCR expression studies in the plant pathogen Pectobacterium atrosepticum
%A Gunnhild W Takle
%A Ian K Toth
%A May B Brurberg
%J BMC Plant Biology
%D 2007
%I BioMed Central
%R 10.1186/1471-2229-7-50
%X Of twelve reference gene candidates tested, four proved to be stably expressed both in six different cultural growth conditions and in planta. Two of these genes (recA and ffh), encoding recombinase A and signal recognition particle protein, respectively, proved to be the most stable set of reference genes under the experimental conditions used. In addition, genes proC and gyrA, encoding pyrroline-5-carboxylate reductase and DNA gyrase, respectively, also displayed relatively stable mRNA expression levels.Based on these results, we suggest recA and ffh as suitable candidates for accurate normalisation of real-time RT-PCR data for experiments investigating the plant pathogen P. atrosepticum and potentially other related pathogens.Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) has become the preferred method for studying low-abundant mRNA expression [1]. The high sensitivity and specificity of RT-PCR makes it a particularly useful and powerful technique for monitoring the mRNA expression of pathogen genes during host infection, where the pathogen's expression profile is often masked by the much higher concentration of host RNA. However, the study of pathogen gene expression inside infected host tissue poses some problems, as there is no straightforward way of measuring the total pathogen RNA concentration. An increase in target transcript at different time points after infection could either come from an up-regulation of transcription or merely from an increase in the pathogen population inside the host tissue, or both. Therefore, normalisation of the data against reference genes (i.e., genes whose expression do not change under the various experimental conditions) is an important step in the quantification of gene expression. Reference genes are also used to correct for differences between samples, such as variation in the total quantity of RNA and variation in RT-PCR efficiency. Therefore, it is of paramount importance to find stably ex
%U http://www.biomedcentral.com/1471-2229/7/50