Evaluation of candidate reference genes in Clostridium difficile for gene expression normalization |
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| Authors: | Devon Metcalf Shayan Sharif J Scott Weese |
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| Institution: | 1. Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI 48109, USA;2. Department of Internal Medicine, Division of Infectious Diseases, University of Michigan, Ann Arbor, MI 48109, USA;3. Division of Infectious Diseases, Department of Medicine, Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA;1. University of Copenhagen, Faculty of Health and Medical Science, Grønnegardsvej 15, 1870 Frederiksberg, Copenhagen, Denmark;2. Technical University of Denmark, National Veterinary Institute, Bülowsvej 27, 1870 Frederiksberg C, Denmark;3. Technical University of Denmark, National Food Institute, Morkhoj Bygade 19, 2860 Søborg, Denmark;4. Clerici-Sacco Group, Via Manzoni 29, 22071 Cadorago, Italy;1. Department of Microbiology and Virology, Faculty of Medicine, Kerman University of Medical Sciences, Kerman, Iran;2. Department of Community Medicine, Faculty of Medicine, Kerman University of Medical Sciences, Kerman, Iran;3. Department of Infectious Diseases, Afzalipour Hospital, Kerman University of Medical Sciences, Kerman, Iran;1. Department of Population Medicine and Diagnostic Sciences, Cornell University, Ithaca, NY 14853, USA;2. Institute of Zoology, Chinese Academy of Sciences, Beijing, China;3. Department of Biomedical Sciences, Cornell University, Ithaca, New York, USA;4. Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, Virginia, USA |
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| Abstract: | Quantitative real-time polymerase chain reaction (qPCR) is a sensitive, efficient and reproducible technique for studying gene expression. Identification of stably expressed reference genes is required to avoid bias in these studies yet mostly unvalidated reference genes are used in studying gene expression in Clostridium difficile. Here, we sought to identify a set of stable reference genes used to normalize C. difficile expression data comparing exponential versus stationary phases of growth. Eight candidate reference genes (rpoA, rrs, gyrA, gluD, adk, rpsJ, tpi, and rho) were assessed in 3 C. difficile genotypes (ribotypes 027, 078, and 001). The primers were analyzed for efficiency and the 8 genes were ranked according to their stability. Overall, the genes rrs, adk, and rpsJ ranked among the most stable. Identification of the most stable genes was, however, strain dependent and suggests that selection of reference genes in a heterogeneous species, such as C. difficile, requires multiple genes to be assessed to confirm their stability within the strains being studied. |
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