Rotator Cuff Injury Muscle RNA Extraction

As previously described (Vasquez-Bolanos et al., 2021 (link)) the muscle samples from the P1 region were removed from −80°C and brought to a cryostat where they were allowed to come up to −20°C. The P1 region was chosen due to consistently presenting the most affected region of muscle in this rotator cuff injury model compared to the anterior and medial regions (Vargas-Vila et al., 2021 (link)). A 50–75 mg piece was removed from the center of each pinned region and placed in a pyrogen-free tube. RNA extraction was performed using the QIAGEN Fibrous Tissue mini kit on a QIAGEN Qiacube robot (QIAGEN, Germantown, MD). In brief, the tissue was immersed in buffer RLT and disrupted by bead in the QIAGEN TissueLyser II, (QIAGEN, Germantown, MD) before being transferred to the Qiacube for RNA extraction. Samples were digested with Proteinase K, (QIAGEN, Germantown, MD) prior to extraction. A DNase digestion step was included in the protocol. RNA was stored at −80°C.