SRA

The context-dependent correlation between mRNA and protein abundance has long been debated. RNA sequencing (RNA-seq), a high-throughput, commonly used method for analyzing transcriptional dynamics and identifying biomarkers, leaves questions about whether we can translate RNA-seq-identified gene signatures directly to protein changes. In this study, we utilized a set of 17 widely assessed immune and wound healing mediators in the context of canine Volumetric Muscle Loss (VML) to investigate the correlation of mRNA and protein abundance. Our data reveal an overall agreement between mRNA and protein levels on these 17 mediators when examining samples from the same experimental condition, such as the same wound biopsy. However, we observed a lack of correlation between mRNA and protein levels for individual genes under different conditions, underscoring the challenges in converting transcript level changes directly into corresponding protein level changes. As an initial attempt to address this discrepancy, we developed a machine-learning model to predict protein abundances from RNA-seq data, achieving high accuracy (Spearman's Rho: 0.78-0.99, imputed versus measured protein abundance; pooling all biopsies; 5-fold cross-validation). Our approach also effectively corrected multiple extreme outliers measured by antibody-based protein assays. Additionally, this model has the potential to detect post-translational modification events, as shown by accurately estimating activated transforming growth factor (TGF)-ß1 levels. While preliminary, this study introduces a promising strategy for translating RNA-seq data into protein abundance and the associated biological relevance. Overall design: The in vivo studies described herein have been approved by the s Institutional Animal Care and Use Committee at the University of Pittsburgh. A VML defect was created in the quadriceps muscles of ten female dogs. Tissue biopsy samples were collected at various intervals from day 1 to 42 following injury. The dogs were prepared for surgery by sedation, shaving of the surgical site, scrubbing with 70% ethyl alcohol, followed by a topical 10% povidone-iodine solution. and induction of surgical plane anesthesia with 2% thiopental sodium, intubation and maintenance with 1-3% isoflurane. A 10 cm long x 4 cm wide area was defined and an initial incision point at the greater trochanter that extended to the lateral epicondyle. The subcutaneous tissue was bluntly dissected and the skin retracted to visualize the biceps femoris. The underlying muscle tissue and fascia were surgically excised to a depth of 2-3 cm. The depth of excision depended upon individual dog anatomy. This procedure results in the removal of between 50-70% of total muscle mass loss. The overlying skin was removed to leave an open wound that mimicked trauma-induced VML in the clinical setting. The upper leg and wound site were covered with sterile dressings and all animals were administered antibiotic prophylaxis for the first 5 days post-surgery (cephalexin, 25 mg/kg) and analgesic every 8-12h for the first 5 days (buprenorphine, 0.002 mg/kg). The overall health of each dog, including temperature, appetite, and activity levels, was monitored daily. The dogs were fed a high-energy, high-protein diet (Advanced Protocol High-Density Canine Diet; PMI Nutrition LLC, Henderson, CO, USA) and provided unlimited access to water.