*2.7. Serum Analysis*

Blood samples were collected serially, either from the jugular vein or abdominal vena cava, at 22 days post-OA induction. Blood samples were centrifuged at 3000 rpm for 12 min to separate the serum. The sera were then stored at −70 ◦C until used for enzyme-linked immunosorbent assay (ELISA) experiments. The serum levels of the pro-inflammatory cytokines tumor necrosis factor alpha (TNFα), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6), and the cartilage degeneration mediators cartilage oligomeric matrix protein (COMP) and C-telopeptide of type II collagen (CTX-II), were determined using commercial ELISA kits (TNFα: cat no. BMS622TEM, Invitrogen, MN, USA; IL-1β: cat no. BMS630; IL-6: cat no. BMS625; COMP: cat no. abx256440, Abbexa Ltd., Cambridge, UK; CTX-II: cat no. E-EL-R2554, Elabscience Biotechnology Inc., Houston, TX, USA) according to manufacturer's protocol, respectively.

#### *2.8. Real-Time PCR Analysis*

Total RNA was extracted from articular cartilage tissues by using QIAzol ® and purified using the miRNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's protocol. Total RNA was reverse transcripted into complementary DNA (cDNA) using a High Capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA, USA), and then subjected to quantitative real-time PCR (qPCR) using QuantiFast ® SYBR Green PCR master mix (Qiagen) with custom-designed specific primers using 18S as house-keeping control on a StepOnePlusTM Real-Time PCR System (Applied Biosystems). The primer sequences are listed in Table 2. Relative fold-changes in target gene expression between groups were determined for all targets using the 2ΔΔCt method.


**Table 2.** List and sequences of real-time PCR primers for mRNA expression.
