Validation of four reference genes for quantitative mRNA expression studies in a rat model of inflammatory injury

Roxanne Y Walder; Anne-Sophie Wattiez; Stephanie R White; Blanca Marquez de Prado; Marta V Hamity; Donna L Hammond

doi:10.1186/1744-8069-10-55

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Validation of four reference genes for quantitative mRNA expression studies in a rat model of inflammatory injury

Journal article

Open access

Peer reviewed

Validation of four reference genes for quantitative mRNA expression studies in a rat model of inflammatory injury

Roxanne Y Walder, Anne-Sophie Wattiez, Stephanie R White, Blanca Marquez de Prado, Marta V Hamity and Donna L Hammond

Molecular pain, Vol.10(1), pp.55-55

09/04/2014

DOI: 10.1186/1744-8069-10-55

PMCID: PMC4161874

PMID: 25187167

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Abstract

Background: Real-time quantitative PCR (qPCR) is a technique frequently used to measure changes in mRNA expression. To ensure validity of experimental findings, it is important to normalize the qPCR data to reference genes that are stable and unaffected by the experimental treatment to correct for variability among samples. Unlike in some models of neuropathic pain, reference genes for models of inflammatory injury have not been validated. This study examined four candidate reference genes in an effort to identify and validate optimal genes for normalization of transcriptional changes occurring in the dorsal horn of the spinal cord and the rostral ventromedial medulla (RVM) following intraplantar injection of complete Freund's adjuvant (CFA). Results: The expression of hypoxanthine phosphoribosyltransferase 1 (Hprt1), beta-actin (Actb), mitogen-activated protein kinase 6 (Mapk6), and beta-2-microglobulin (B2m) was quantified in the dorsal horn and RVM of rats four days or two weeks after intraplantar injection of CFA or saline. The range of expression levels among these four genes differed by as much as 16-fold within the dorsal horn and the RVM. All four of these reference genes were stably expressed in both tissues and did not differ between saline and CFA-treated animals. Analyses using the statistical algorithms in geNorm and NormFinder programs determined that Mapk6 was the most stable gene and recommended the combination of Mapk6 and Actb, or Mapk6 and Hprt1, in such experimental conditions. Conclusions: This study validated the four genes Hprt1, Actb, Mapk6 or B2m and showed that any one or combination of two of them are good reference genes for normalization of mRNA expression in qPCR experiments in the spinal cord and RVM in the CFA model of inflammatory injury.

Pain

Nociception

Housekeeping genes

Research

Complete Freund’s adjuvant

Rostral ventromedial medulla

Hprt1

Mapk6

B2m

Actb

Dorsal horn

Details

Title: Subtitle: Validation of four reference genes for quantitative mRNA expression studies in a rat model of inflammatory injury
Creators: Roxanne Y Walder - Departments of Anesthesia, University of Iowa, Iowa City, IA 52242 USA
Anne-Sophie Wattiez - Departments of Anesthesia, University of Iowa, Iowa City, IA 52242 USA
Stephanie R White - University of Iowa
Blanca Marquez de Prado - Departments of Anesthesia, University of Iowa, Iowa City, IA 52242 USA
Marta V Hamity - Departments of Anesthesia, University of Iowa, Iowa City, IA 52242 USA
Donna L Hammond - Departments of Anesthesia, University of Iowa, Iowa City, IA 52242 USA
Resource Type: Journal article
Publication Details: Molecular pain, Vol.10(1), pp.55-55
DOI: 10.1186/1744-8069-10-55
PMID: 25187167
PMCID: PMC4161874
NLM abbreviation: Mol Pain
ISSN: 1744-8069
eISSN: 1744-8069
Publisher: BioMed Central; London
Language: English
Date published: 09/04/2014
Academic Unit: Molecular Physiology and Biophysics; Iowa Neuroscience Institute; Nursing; Anesthesia; Neuroscience and Pharmacology; Internal Medicine
Record Identifier: 9984006478502771

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