Quantification of gene expression after painful nerve injury: validation of optimal reference genes. J Mol Neurosci 2012 Mar;46(3):497-504
Date
08/25/2011Pubmed ID
21863315Pubmed Central ID
PMC3273664DOI
10.1007/s12031-011-9628-xScopus ID
2-s2.0-84860411132 (requires institutional sign-in at Scopus site) 38 CitationsAbstract
Stably expressed housekeeping genes (HKGs) are necessary for standardization of transcript measurement by quantitative real-time polymerase chain reaction (qRT-PCR). Peripheral nerve injury disrupts expression of numerous genes in sensory neurons, but the stability of conventional HKGs has not been tested in this context. We examined the stability of candidate HKGs during nerve injury, including the commonly used 18S ribosomal RNA, β-tubulin I and β-tubulin III, actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and hypoxanthine phosphoribosyltransferase 1 (HPRT1), and mitogen-activated protein kinase 6 (MAPK6). Total RNA for cDNA synthesis was isolated from dorsal root ganglia of rats at 3, 7, and 21 days following either skin incision alone or spinal nerve ligation, after which the axotomized and adjacent ganglia were analyzed separately. Relative stability of HKGs was determined using statistical algorithms geNorm and NormFinder. Both analyses identified MAPK6 and GAPDH as the two most stable HKGs for normalizing gene expression for qRT-PCR analysis in the context of peripheral nerve injury. Our findings indicate that a prior analysis of HKG expression levels is important for accurate normalization of gene expression in models of nerve injury.
Author List
Bangaru ML, Park F, Hudmon A, McCallum JB, Hogan QHAuthor
Quinn H. Hogan MD Professor in the Anesthesiology department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
AnimalsDisease Models, Animal
Gene Expression Regulation, Enzymologic
Gene Targeting
Genes, Essential
Male
Membrane Glycoproteins
Mitogen-Activated Protein Kinase 6
Neuralgia
Proteins
Rats
Rats, Sprague-Dawley
Reference Standards
Sensory Receptor Cells
Stromal Interaction Molecule 1
