Identification and validation of suitable housekeeping genes for normalizing quantitative real-time PCR assays in injured peripheral nerves

Giovanna Gambarotta; Giulia Ronchi; Olivier Friard; Pantaleo Galletta; Isabelle Perroteau; Stefano Geuna

doi:10.1371/journal.pone.0105601

Identification and validation of suitable housekeeping genes for normalizing quantitative real-time PCR assays in injured peripheral nerves

PLoS One. 2014 Aug 21;9(8):e105601. doi: 10.1371/journal.pone.0105601. eCollection 2014.

Authors

Giovanna Gambarotta¹, Giulia Ronchi², Olivier Friard³, Pantaleo Galletta⁴, Isabelle Perroteau¹, Stefano Geuna⁵

Affiliations

¹ Department of Clinical and Biological Sciences, University of Torino, Orbassano, Italy; Neuroscience Institute of Turin (NIT), University of Torino, Orbassano, Italy.
² Department of Clinical and Biological Sciences, University of Torino, Orbassano, Italy; Neuroscience Institute of the "Cavalieri Ottolenghi" Foundation (NICO), University of Torino, Orbassano, Italy.
³ Department of Life Sciences and Systems Biology, University of Torino, Torino, Italy.
⁴ Department of Clinical and Biological Sciences, University of Torino, Orbassano, Italy.
⁵ 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.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Disease Models, Animal
Female
Gene Expression Profiling
Genes, Essential*
Peripheral Nerve Injuries / diagnosis
Peripheral Nerve Injuries / genetics*
RNA Stability
Rats
Real-Time Polymerase Chain Reaction*
Reproducibility of Results

Grants and funding

The research leading to this paper has received funding from the European Community's Seventh Framework Programme (FP7-HEALTH-2011) under grant agreement no. 278612 (BIOHYBRID), from MIUR and from Compagnia di San Paolo (MOVAG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.