The equine carpal osteochondral fragment model
The equine carpal osteochondral fragment model
Synovial fluid samples were obtained from both middle carpal joints prior to (day 0), day 28  and 70 days after surgery. Horses were sedated with detomidine (0.01 mg/kg, Domosedan Vet, Orion Pharma Animal Health, Copenhagen, Denmark) and butorphanol tartrate (0.01 mg/kg, Dolorex, MSD Animal Health, Copenhagen, Denmark), and the dorsal surface of the carpi prepared aseptically by chlorhexidine scrubbing and alcohol swabbing. Synovial fluid was aspirated using a 19 gauge 40 mm needle and immediately transferred into ethylene diamine tetra acetic acid (EDTA) tubes (BD Vacutainer, BD A/S, Albertslund, Denmark).  Tubes were inverted carefully 5-10 times and samples were further processed as detailed in Table 1 within one hour and stored at -80 ‏⁰C until small RNA sequencing and RT-qPCR analysis of miRNA expression (performed within 6 months).  500 µl SF were treated with a 1ug/µl hyaluronidase (HA) solution to reduce viscosity as preciously described [12]. RNA was extracted from SF using miRNeasy serum/plasma Advanced Kit (Qiagen, Vedbæk, Denmark) according to the manufacturer's instructions. Samples underwent on-column DNase digestion with RNase free DNase sets (Qiagen, Vedbæk, Denmark) and RNA yield was determined by spectrophotometry (Nano Drop ND-1000 spectrophotometer, Saveen and Werner AB, Limhamn, Sweeden). This project utilized the NEBNext® Small RNA Library Prep Set for Illumina® (Multiplex Compatible). The protocol was adapted to accommodate the low input of sample by dilution of adapters 1 in 5 and extra PCR cycles.