Suitable reference gene selection for gene expression studies in knee osteoarthritis synovium using quantitative PCR analysis

Osteoarthritis (OA) is the leading cause of musculoskeletal disability in the elderly. Insights into the biological features of OA are obtained by characterization of the molecular features by gene expression profiling using reverse transcription-quantitative PCR (RT-qPCR). However, it has recently become evident that the use of suitable reference genes is required for appropriate normalization of this technique. Here total RNA was isolated from the synovium of 18 men and 20 women who underwent total knee arthroplasty for knee OA (KOA). We validated the expression stability of 7 candidate housekeeping genes (<i>ACTB, B2M, GAPDH, HPRT1, RPL13A, SDHA</i>, and <i>YWHAZ</i>) in the synovium of KOA with 3 commonly used algorithms (geNorm, NormFinder, and BestKeeper). Additionally, we evaluated expression profiles of the steroid hormone receptor (<i>AR, ESR1, ESR2, GR, MR</i>, and <i>PR</i>) and proinflammatory cytokines (<i>IL1B</i> and <i>IL6</i>) genes in the synovium and their correlations with the risk factors of KOA, using the most and least stable housekeeping genes for comparison. Results showed that <i>HPRT1</i> was the most stable gene, whereas <i>B2M</i> was the least stable. RT-qPCR analysis revealed sexually dimorphic expression of <i>AR, IL1B</i>, and <i>IL6</i>; intercorrelations between steroid hormone receptor expression levels and female-specific correlations of <i>IL1B</i> expression with <i>ESR1</i> and <i>PR</i> expression, <i>IL6</i> expression with <i>ESR1</i> and <i>GR</i> expression, and body mass index with <i>AR</i> and <i>PR</i> expression; and the choice of the least stable reference gene altered several correlations and statistical significances. In conclusion, <i>HPRT1</i> was identified as the suitable reference gene for normalization in the OA synovium.