Background

Osteoarthritis (OA) is the prevailing cause of joint impairment, particularly in the knee joint, and is characterized primarily by inflammation, redness in the affected joints, persistent joint pain, and restricted movement. Based on the Global Burden of Disease Study 2019, research from 1990 to 2019 revealed a substantial increase in the prevalence of OA in specific joints. The prevalence of OA cases increased significantly by 113%, increasing from 247 million in 1990 to 527 million in 2019, with knee OA playing a major role in contributing to the overall burden [1]. Therefore, there is a compelling need to gain deeper insights into the pathological mechanisms underlying OA, and novel approaches, methodologies, and techniques must be employed to elucidate the pathogenesis of OA and to further explore therapeutic strategies. Articular cartilage, which is the most affected tissue in osteoarthritic conditions and undergoes degenerative disease, has a distinctive structural composition and consists of a very sparse distribution of chondrocytes surrounded by copious amounts of extracellular matrix (ECM) [2]. The majority of articular cartilage is composed of ECM, which consists mainly of glycosaminoglycans, collagens, and proteoglycans [3]. Moreover, the gradual deterioration of the articular surfaces in OA results in a significant decrease in the amount of cartilage and, consequently, in the amount of available tissue. This adds to the existing challenges faced while isolating RNA from cartilage tissues and underscores the importance of devising an appropriate methodology for procuring high-quality RNA from such tissues. In addition, articular cartilage is relatively hard, making it even more difficult to homogenize for RNA extraction without specialized equipment. To date, some of the reported methods for isolating RNA from articular cartilage are suitable only for larger cartilage samples weighing at least 100 mg, or they require the use of freezer-mills or microdisembranators [4,5] for homogenization, which are not readily available in all laboratories. The technique of RNA extraction from cartilage by first isolating chondrocytes [6,7] has certain drawbacks, as it can lead to the loss of heterogeneous chondrocyte populations in the cartilage and can be stressful for the cells, leading to a skewed representation of the gene expression profile. In addition, this is also a time-consuming process, especially when dealing with a large number of samples, which can lead to delays in downstream analysis. Protocols for the isolation of RNA from the cartilage of humans or large animals (e.g., chickens and horses) [8,9] are available, but this is not the case for Dunkin–Hartley guinea pigs, despite being the most appropriate model for studying idiopathic osteoarthritis [10–12]. While studies in smaller species such as mice often achieve high RNA quality, they frequently require pooling cartilage tissue from multiple animals to obtain sufficient material for extraction [13]. Moreover, advanced techniques such as single-cell RNA sequencing in mice also necessitate pooling cartilage from a minimum of five mice [14], which may not be feasible or align with certain experimental designs, especially in contexts where animal availability is restricted.

Owing to its limited cellular population and elevated proteoglycan content, the extraction of high-quality total RNA from cartilage poses a particular challenge. This makes gene expression analysis directly from the cartilage tissues technically demanding, yet critically important. High-quality RNA is essential not only for accurate gene expression profiling but also for exploring epigenetic mechanisms involved in OA, such as regulation by non-coding RNAs (e.g., microRNAs). Gene expression analysis provides a molecular snapshot of the disease state and allows for the identification of differentially expressed genes and signaling pathways involved in OA progression, inflammation, and cartilage degradation. These molecular signatures can help uncover key regulatory nodes that drive disease onset and advancement, thereby serving as targets for therapeutic intervention. While gene expression alone may not fully elucidate OA pathogenesis, it remains a foundational and indispensable tool in dissecting the molecular mechanisms of the disease and guiding the development of targeted, mechanism-based therapies.

Based on the importance of investigating gene expression within chondrocytes, the challenges associated with RNA isolation from small amounts of cartilage tissue, and the limitations of existing methodologies, we present an improved RNA isolation protocol in resource-limited setups from the articular cartilage and synovium of Dunkin–Hartley guinea pigs. The protocol has been designed to optimize sample collection and tissue processing procedures, aiming to minimize RNA degradation while increasing RNA quality. It has significant implications for advancing both basic OA research and translational applications. Our findings demonstrate that the RNA obtained from the articular cartilage and synovial tissues of Dunkin–Hartley guinea pigs using the optimized methodology is suitable for downstream applications such as RTqPCR. However, a few limitations remain. While the extracted RNA exhibited acceptable purity and concentration, the RNA integrity number (RIN) values were below the threshold generally required for high-throughput applications such as RNA sequencing or transcriptome profiling. Despite this, the RNA quality was sufficient for reliable RT–qPCR analysis, providing a practical and cost-effective alternative, particularly for resource-limited settings. Additionally, we observed variability in RNA yield from 12-month-old animals, likely due to increased OA severity and the corresponding reduction in the quantity of available tissue. This variability poses a challenge for reproducibility and consistency. Future refinements in tissue handling, preservation, and extraction techniques may be necessary to further enhance RNA quality and expand the applicability of this protocol to broader transcriptomic studies.