Background: Osteoarthritis (OA) is the primary cause of joint impairment, particularly in the knee. The prevalence of OA has significantly increased, with knee OA being a major contributor whose pathogenesis remains unknown. Articular cartilage and synovium play critical roles in OA, but extracting high-quality RNA from these tissues is challenging due to high extracellular matrix content and low cellularity. This study aimed to identify the most suitable RNA isolation method for obtaining high-quality RNA from micro-quantities of guinea pig cartilage and synovium tissues, a relevant model for idiopathic OA.
Methods and Results: We compared the traditional Trizol® method with modifications against spin-column-based methods (TRIspin -Trizol®/ RNeasy™, RNeasy™ kit, RNAqueous™ kit, Quick-RNA™ Miniprep Plus Kit) and an optimized RNA isolation protocol was developed to enhance RNA yield and purity. The procedure involved meticulous sample collection, specialized tissue processing, and measures to minimize RNA degradation. RNA quality was assessed using spectrophotometry and RT-qPCR. The results demonstrated that among the tested methods, the Quick-RNA™ Miniprep Plus Kit with proteinase K treatment provided the highest quality RNA from limited cartilage and synovium samples.
Conclusions: Preparation, storage, homogenization, and the method of RNA isolation are critical for obtaining high-purity RNA from guinea pig cartilage and synovium tissues. Our developed protocol significantly enhances RNA quality and purity, making it particularly effective for RT-qPCR and gene expression studies in osteoarthritis research using small animal models like Dunkin-Hartley guinea pigs. Further refinements can potentially enhance RNA yield and purity but this protocol facilitates more accurate molecular analyses, contributing to a better understanding of OA pathogenesis and the development of therapeutic strategies.
