59
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      Evaluation of reference genes for real-time RT-PCR expression studies in the plant pathogen Pectobacterium atrosepticum

      research-article
      1 , 2 , 3 , 1 ,
      BMC Plant Biology
      BioMed Central

      Read this article at

      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          Background

          Real-time RT-PCR has become a powerful technique to monitor low-abundance mRNA expression and is a useful tool when examining bacterial gene expression inside infected host tissues. However, correct evaluation of data requires accurate and reliable normalisation against internal standards. Thus, the identification of reference genes whose expression does not change during the course of the experiment is of paramount importance. Here, we present a study where manipulation of cultural growth conditions and in planta experiments have been used to validate the expression stability of reference gene candidates for the plant pathogen Pectobacterium atrosepticum, belonging to the family Enterobacteriaceae.

          Results

          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.

          Conclusion

          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.

          Related collections

          Most cited references45

          • Record: found
          • Abstract: not found
          • Book: not found

          Molecular Cloning : A Laboratory Manual

            Bookmark
            • Record: found
            • Abstract: not found
            • Article: not found

            The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normalization.

              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              Unravelling the biology of macrophage infection by gene expression profiling of intracellular Salmonella enterica.

              For intracellular pathogens such as Salmonellae, Mycobacteriae and Brucellae, infection requires adaptation to the intracellular environment of the phagocytic cell. The transition from extracellular to intravacuolar environment has been expected to involve a global modulation of bacterial gene expression, but the precise events have been difficult to determine. We now report the complete transcriptional profile of intracellular Salmonella enterica sv. Typhimurium following macrophage infection. During replication in murine macrophage-like J774-A.1 cells, 919 of 4451 S. Typhimurium genes showed significant changes in transcription. The expression profile identified alterations in numerous virulence and SOS response genes and revealed unexpected findings concerning the biology of the Salmonella-macrophage interaction. We observed that intracellular Salmonella are not starved for amino acids or iron (Fe2+), and that the intravacuolar environment is low in phosphate and magnesium but high in potassium. S. Typhimurium appears to be using the Entner-Douderoff pathway to use gluconate and related sugars as a carbon source within macrophages. Almost half the in vivo-regulated genes were of unknown function, suggesting that intracellular growth involves novel macrophage-associated functions. This is the first report that identifies the whole set of in vivo-regulated genes for any bacterial pathogen during infection of mammalian cells.
                Bookmark

                Author and article information

                Journal
                BMC Plant Biol
                BMC Plant Biology
                BioMed Central
                1471-2229
                2007
                21 September 2007
                : 7
                : 50
                Affiliations
                [1 ]Norwegian Institute for Agricultural and Environmental Research, Plant Health and Plant Protection Division, Høgskoleveien 7, 1432 Ås, Norway
                [2 ]Norwegian University of Life Sciences, Institute for Chemistry, Biotechnology and Food Science, PO Box 5003, 1432 Ås, Norway
                [3 ]SCRI, Invergowrie, Dundee DD2 5DA, UK
                Article
                1471-2229-7-50
                10.1186/1471-2229-7-50
                2151947
                17888160
                263faf35-358d-4c09-8a55-6aaf3fac89c9
                Copyright © 2007 Takle et al; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 13 February 2007
                : 21 September 2007
                Categories
                Research Article

                Plant science & Botany
                Plant science & Botany

                Comments

                Comment on this article