Peripheral blood (PB) collection and miR-29cb2 detection

PB (2 mL) remaining from preoperative routine blood tests of the patients who underwent joint-related surgery were included. To prevent RNA degradation, whole blood was collected into PAXgene Blood RNA tubes for subsequent detection. Then, total RNA was concentrated and purified using a PAXgene Blood RNA kit. In brief, the pellet collected from the initial centrifugation was incubated in optimized buffers with proteinase K to digest proteins. After incubation at 55°C for 10 min, the suspension was transferred to a PAXgene Shredder spin column (PSC) to homogenize the cell lysate and remove residual cell debris. After adding ethanol to the aforementioned supernatant to adjust the binding conditions, the lysate was applied to a PAXgene RNA spin column (PRC). During a brief centrifugation step, the RNA was selectively bound to the silica membrane of the PAXgene column as contaminants passed through. The remaining contaminants were removed through several efficient wash steps. Between the first and second wash steps, the membrane was treated with DNase I (RNFD) to remove trace amounts of bound DNA. After the wash steps, RNA was eluted in elution buffer and heat denatured, it was then ready for subsequent detection. miR-29 family members were detected by RT-PCR as described above.