Abstract Body: Background
Chronic Diabetic Foot Ulcers (DFUs) can lead to complications such as lower-extremity amputations, if left untreated. It is not known why some patients respond to standard of care treatment while others do not. The goal of this study was to search for a optimal RNA extraction protocol and understand the influence of patient factors on wound pathology, as measured by whole transcriptome analysis. This information will lead to identifying a clinical biomarker of wound inflammation, which would allow patients to receive treatments in a personalized fashion.

Methods
Debrided DFU tissue samples from patients undergoing treatment at Northwell Hospital in NY were used. Inclusion criteria included being 18+ years of age with a diagnosis of type 2 diabetes and an ulcer that had been open for at least 8 weeks at the time of enrollment. Clinical factors collected include race, sex, age, body mass index, comorbidities, and smoking status. Samples were classified as healed or non-healed at 12 weeks from sample collection. Samples were categorized as younger (<60 years, n=13) and older (>75 years, n=13). Total RNA was extracted from debrided DFU tissue stored in RNA later. The RNA integrity number (RIN), as well as RNA yield and analyzing fragment size distribution (DV200) were measured by Agilent 2100 BioAnalyzer. RNA purity (OD 260/280 ratio) was measured using a Nanodrop. >200 ng of total RNA, DV200 > 3 and OD 260/280 ratio > 7 the minimum quality control (QC) metrics were considered.

Results
After total RNA extraction from debrided DFUs, the purity, integrity, and quality of RNA were assessed. Sample yield values ranged from 3.6 to 2,000 ng, indicating that out of 26 samples, 16 met the minimum QC thresholds. RIN and DV200 analysis confirmed that only 14 out of 16 samples were according to the minimum RNA integrity requirement. 260/280nm ratio ranged from 1.7 to Y 2.2 nm, indicating that all 14 samples met the minimum QC thresholds.

Conclusion
Based on these results, we conclude that using our RNA-extraction protocol, we can obtain RNA from DFU wound samples according to minimum QC thresholds. Of Isolated high-quality RNA 14 patients (8 young and 6 old) all are non-healing and all clinical factors are the same except for age. Therefore, we will evaluate the influence of age on wound transcriptomes. Understanding the cellular mechanisms underlying non-healing DFUs and specific inflammatory pathways will provide insight for targeted treatment.