Quantification of gene expression after painful nerve injury

Validation of optimal reference genes

Madhavi Latha Yadav Bangaru, Frank Park, Andy Hudmon, J. Bruce McCallum, Quinn H. Hogan

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)497-504
Number of pages8
JournalJournal of Molecular Neuroscience
Volume46
Issue number3
DOIs
StatePublished - Mar 1 2012
Externally publishedYes

Fingerprint

Essential Genes
Gene Expression
Wounds and Injuries
Mitogen-Activated Protein Kinase 6
Genes
Peripheral Nerve Injuries
Glyceraldehyde-3-Phosphate Dehydrogenases
Tubulin
Real-Time Polymerase Chain Reaction
18S Ribosomal RNA
Hypoxanthine Phosphoribosyltransferase
Spinal Nerves
Spinal Ganglia
Sensory Receptor Cells
Ganglia
Ligation
Actins
Complementary DNA
RNA
Skin

All Science Journal Classification (ASJC) codes

  • Cellular and Molecular Neuroscience

Cite this

Quantification of gene expression after painful nerve injury : Validation of optimal reference genes. / Bangaru, Madhavi Latha Yadav; Park, Frank; Hudmon, Andy; McCallum, J. Bruce; Hogan, Quinn H.

In: Journal of Molecular Neuroscience, Vol. 46, No. 3, 01.03.2012, p. 497-504.

Research output: Contribution to journalArticle

Bangaru, Madhavi Latha Yadav ; Park, Frank ; Hudmon, Andy ; McCallum, J. Bruce ; Hogan, Quinn H. / Quantification of gene expression after painful nerve injury : Validation of optimal reference genes. In: Journal of Molecular Neuroscience. 2012 ; Vol. 46, No. 3. pp. 497-504.
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