Abstract
Real time quantitative PCR (RT-qPCR) requires a method to normalize the expression of target genes against an en- dogenous reference gene. It is known that commonly used housekeeping genes (HKGs) vary tremendously in inflam- matory conditions; however information about the stability and expression of HKGs in chronic inflammatory joint dis- ease 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 glyce-raldehyde-3-phosphate dehydrogenase (GAPDH) was found as the least stable gene in musculoskeletal tissues. In in-flamed 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 refer- ence 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.
Highlights
Real time quantitative PCR (RT-qPCR) is a sensitive and reproducible method for the estimation and comparative analysis of mRNA expression [1]
10 housekeeping genes (HKGs) were run in the neuronal (SC, dorsal root ganglia (DRG)) and in the musculoskeletal tissues from the control and arthritic rats (Table 1)
18S was excluded in DRG, B2M in muscle, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and UCE in capsule, GAPDH in epiphysis and GUSB, peptidylprolyl isomerase B (PPIB), ACTB and ARBP in periosteum (Table 2)
Summary
Real time quantitative PCR (RT-qPCR) is a sensitive and reproducible method for the estimation and comparative analysis of mRNA expression [1]. It requires a method to normalize the expression of target genes against an endogenous reference or housekeeping gene (HKG) to see the difference in RNA concentration. A prerequisite for a gene to serve as endogenous reference is that its expression level should not vary in different tissue types and under various experimental conditions. Previous studies indicate that most commonly used HKGs i.e., glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin (ACTB) show considerable variation between tissues as well as in different experimental conditions [2]. Little information is available about the expression of HKGs in chronic inflammatory joint diseases such as rheumatoid arthritis (RA). It seems reasonable to speculate that inflammatory cell infiltration can cause huge differences in the level of expression of HKGs genes leading to erroneous results. Appropriate validation of HKGs is a crucial step in inflammatory conditions
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