title: Validation of four reference genes for quantitative mRNA expression studies in a rat model of inflammatory injury.
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.
authors.forename: Roxanne Y
authors.initials: RY
authors.lastname: Walder
authors.forename: Anne-Sophie
authors.initials: AS
authors.lastname: Wattiez
authors.forename: Stephanie R
authors.initials: SR
authors.lastname: White
authors.forename: Blanca
authors.initials: B
authors.lastname: Marquez de Prado
authors.forename: Marta V
authors.initials: MV
authors.lastname: Hamity
authors.affiliation: Departments of Anesthesia, University of Iowa, Iowa City, IA 52242, USA. donna-hammond@uiowa.edu.
authors.forename: Donna L
authors.initials: DL
authors.lastname: Hammond
mesh_terms: D000199:Actins / Q000235:genetics* / Q000378:metabolism; D000465:Algorithms; D000818:Animals; D004195:Disease Models, Animal; D005620:Freund's Adjuvant / Q000633:toxicity; D015870:Gene Expression / Q000187:drug effects; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics* / Q000378:metabolism; D007249:Inflammation / Q000139:chemically induced / Q000378:metabolism* / Q000473:pathology; D008297:Male; D008526:Medulla Oblongata / Q000187:drug effects / Q000378:metabolism; D048053:Mitogen-Activated Protein Kinase 6 / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012984:Software; D013116:Spinal Cord / Q000187:drug effects / Q000378:metabolism; D001613:beta 2-Microglobulin / Q000235:genetics* / Q000378:metabolism
chemical_list: D000199:Actins; D012333:RNA, Messenger; D001613:beta 2-Microglobulin; D005620:Freund's Adjuvant; D007041:Hypoxanthine Phosphoribosyltransferase; D048053:Mitogen-Activated Protein Kinase 6