PBMC isolation and RNA extraction
Ficoll density gradient centrifugation was used to collect PBMCs from AS and control patient blood samples using room temperature Lymphoprep (Stemcell, USA). Next, a MiRNeasy Mini Kit (Qiagen, Germany) was utilized for extraction of total RNA from these cells which was then purified, quantified, and assessed for quality as discribed in a prior study[23].