Determination of the most suitable reference gene for transcription analysis of synovial tissue from osteoarthritis patients

Abstract

Abstract Synovial tissue plays a key role in osteoarthritis (OA) pathogenesis. Gene expression analysis is widely used to investigate underlying pathomechanisms; however, accurate normalization of target mRNA expression depends on the use of stable reference genes. This study evaluated the suitability of common reference genes for determining synovial tissue mRNA expression, considering various factors. In the synovial tissue of 20 patients with end-stage OA, the stability of the expression of 10 reference genes ( GAPDH , RPLP0 , YWHAZ , TBP , PPIA , EEF1A1 , ACTB , HRPT1 , SDHA , and RPL13A ) was evaluated using four different analysis methods (NormFinder, geNorm, the ΔCt method, and Pearson correlation analysis). The most stable genes were identified by means of a subsequent ranking analysis. Both the entire sample pool and various subgroups (sex, age, BMI, and synovitis score) were considered. RPL13A , PPIA , and EEF1A1 were identified as the most stable reference genes overall, with minor variability across subgroups. In contrast, GAPDH proved to be the least suitable reference gene, showing the most variable expression. The stability of the reference gene might be affected by sex, age, and BMI and this should be taken into account.