title: Identification and validation of suitable housekeeping genes for normalizing quantitative real-time PCR assays in injured peripheral nerves.
abstract: Injury to the peripheral nerve induces dramatic changes in terms of cellular composition that are reflected on RNA quality and quantity, making messenger RNA expression analysis very complex. Several commonly used housekeeping genes are regulated following peripheral nerve injury and are thus not suitable for quantitative real-time PCR normalization; moreover, the presence of pseudogenes in some of them impairs their use. To deal with this problem, we have developed a new method to identify new stable housekeeping genes based on publicly available microarray data on normal and injured nerves. Four new candidate stable genes were identified and validated by quantitative real-time PCR analysis on nerves during the different phases after nerve injury: nerve degeneration, regeneration and remyelination. The stability measure of these genes was calculated with both NormFinder and geNorm algorithms and compared with six commonly used housekeeping genes. This procedure allowed us to identify two new and highly stable genes that can be employed for normalizing injured peripheral nerve data: ANKRD27 and RICTOR. Besides providing a tool for peripheral nerve research, our study also describes a simple and cheap procedure that can be used to identify suitable housekeeping genes in other tissues and organs.
authors.affiliation: Department of Clinical and Biological Sciences, University of Torino, Orbassano, Italy; Neuroscience Institute of Turin (NIT), University of Torino, Orbassano, Italy.
authors.forename: Giovanna
authors.initials: G
authors.lastname: Gambarotta
authors.affiliation: Department of Clinical and Biological Sciences, University of Torino, Orbassano, Italy; Neuroscience Institute of the "Cavalieri Ottolenghi" Foundation (NICO), University of Torino, Orbassano, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Ronchi
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, Torino, Italy.
authors.forename: Olivier
authors.initials: O
authors.lastname: Friard
authors.affiliation: Department of Clinical and Biological Sciences, University of Torino, Orbassano, Italy.
authors.forename: Pantaleo
authors.initials: P
authors.lastname: Galletta
authors.affiliation: Department of Clinical and Biological Sciences, University of Torino, Orbassano, Italy; Neuroscience Institute of Turin (NIT), University of Torino, Orbassano, Italy.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Perroteau
authors.affiliation: Department of Clinical and Biological Sciences, University of Torino, Orbassano, Italy; Neuroscience Institute of Turin (NIT), University of Torino, Orbassano, Italy; Neuroscience Institute of the "Cavalieri Ottolenghi" Foundation (NICO), University of Torino, Orbassano, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Geuna
mesh_terms: D000818:Animals; D004195:Disease Models, Animal; D005260:Female; D020869:Gene Expression Profiling; D020043:Genes, Essential; D059348:Peripheral Nerve Injuries / Q000175:diagnosis / Q000235:genetics*; D020871:RNA Stability; D051381:Rats; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results