Validation of Reference Genes for mRNA Quantification in Adjuvant Arthritis

Real time quantitative PCR (RT-qPCR) requires a method to normalize the expression of target genes against an endogenous reference gene. It is known that commonly used housekeeping genes (HKGs) vary tremendously in inflammatory conditions; however information about the stability and expression of HKGs in chronic inflammatory joint disease such as rheumatoid arthritis (RA) is scarce. The expressional stability of 10 commonly used HKGs was analyzed in the neuronal (spinal cord, dorsal root ganglia) and in the musculoskeletal tissues (tendon, muscle, epiphysis, capsule, periosteum and ankle joint) using RT-qPCR in the rat model of RA. In individual tissues, suitable HKGs were selected by Ct  (│Ct control − Ct arthritis│) and further analyzed by using software programs; geNorm and normfinder. We found hypoxanthine-guanine phosphoribosyl tranferase (HPRT) as the most stable gene except ankle joint while glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was found as the least stable gene in musculoskeletal tissues. In inflamed ankle joint where no reference gene was found to be stably expressed, an inflammatory cell marker CD3 was used to normalize peptidylprolyl isomerase B (PPIB), the most homogenous HKG identified among the 10 HKGs. The normalized PPIB was then used to analyze the gene expression of neurokinin 1 (NK1), receptor of substance P, a potent pro-inflammatory mediator. We observed a 3.5 fold increase (p = 0.009) in NK1 expression in inflamed ankle joint compared to control. Our results indicate that reference genes stability should be evaluated before using them as reference during inflammatory conditions. In tissues with intense inflammatory cell infiltration, an inflammatory cell marker should be used to normalize the selected reference gene to avoid erroneous results.