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

Hiroshi Watanabe
Hirotaka Ishii
Kenji Takahashi
Shinro Takai
Hitoshi Ozawa

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 (ACTB
 B2M
 GAPDH
 HPRT1
 RPL13A
 SDHA
 and YWHAZ) in the synovium of KOA with 3 commonly used algorithms (geNorm
 NormFinder
 and BestKeeper). Additionally
 we evaluated expression profiles of the steroid hormone receptor (AR
 ESR1
 ESR2
 GR
 MR
 and PR) and proinflammatory cytokines (IL1B and IL6) 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 HPRT1 was the most stable gene
 whereas B2M was the least stable. RT-qPCR analysis revealed sexually dimorphic expression of AR
 IL1B
 and IL6; intercorrelations between steroid hormone receptor expression levels and female-specific correlations of IL1B expression with ESR1 and PR expression
 IL6 expression with ESR1 and GR expression
 and body mass index with AR and PR expression; and the choice of the least stable reference gene altered several correlations and statistical significances. In conclusion
 HPRT1 was identified as the suitable reference gene for normalization in the OA synovium.

Connective tissue research

TAYLOR & FRANCIS INC

59

4

10.1080/03008207.2017.1391234

https://doi.org/10.1080/03008207.2017.1391234
https://www.ncbi.nlm.nih.gov/pubmed/29095075
https://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=JSTA_CEL&SrcApp=J_Gate_JST&DestLinkType=FullRecord&KeyUT=WOS:000438108400006&DestApp=WOS_CPL
http://europepmc.org/abstract/med/29095075
http://orcid.org/0000-0003-2044-316X