In the analysis, human cartilage tissues were precisely divided into cartilage zones using an LCM device (PixCell IIe; Arcturus, Mountain View, CA, USA), and the gene expression was determined in respective zones. Cartilage obtained from 16 OA knees and 9 control knees was used for this analysis. In OA knees, cartilage samples were harvested from both macroscopically intact areas (preserved areas) and areas showing various degrees of cartilage degeneration (degenerated areas). In control knees, cartilage samples were obtained at 1–3 sites in each knee from the femoral condyles, confirming that the areas showed little sign of cartilage degeneration. At each site, approximately 20 mm × 5 mm of cartilage was obtained in full thickness above the tide mark. The tissue was immediately embedded in OCT compound (Sakura Finetek Japan, Tokyo, Japan), snap-frozen in liquid nitrogen, and stored at -80 °C.
For analyses, cryosections were prepared from the OCT-embedded cartilage tissues, and cartilage zones were separated by LCM as described previously [3 (link), 20 (link)]. In detail, the cryosections were cut into 20- to 40-mm-thick slivers, and first treated with 0.5 M EDTA (pH 8.0) for 3 minutes, before being dehydrated with graded concentrations of ethanol, and clarified with xylene. All reagents were RNase-free, and the entire process was completed within 30 minutes to minimize RNA degradation. The sections were then placed on glass slides, set on an LCM device, and divided into cartilage zones by LCM based on their histological features [22 ] (Fig.1 ). Immediately after LCM, RNA was extracted from the respective cartilage zones using an RNeasy Micro kit (Qiagen GmbH, Hiden, Germany) with the routine use of DNase I (Qiagen). cDNA was synthesized using Sensiscript reverse transcriptase (Qiagen). The gene expression was evaluated quantitatively by qPCR on a LightCycler (Roche Diagnostics, Basel, Switzerland), using gene-specific primers and probes. SYBR® Premix Ex Taq® Perfect Real Time (Takara Bio, Shiga, Japan) or Premix Ex Taq® Perfect Real Time (Takara Bio) was used for PCR. The cDNA levels were normalized by the expression of GAPDH.
For analyses, cryosections were prepared from the OCT-embedded cartilage tissues, and cartilage zones were separated by LCM as described previously [3 (link), 20 (link)]. In detail, the cryosections were cut into 20- to 40-mm-thick slivers, and first treated with 0.5 M EDTA (pH 8.0) for 3 minutes, before being dehydrated with graded concentrations of ethanol, and clarified with xylene. All reagents were RNase-free, and the entire process was completed within 30 minutes to minimize RNA degradation. The sections were then placed on glass slides, set on an LCM device, and divided into cartilage zones by LCM based on their histological features [22 ] (Fig.
