Droplet digital PCR (ddPCR) is a novel and improved molecular method that allows for absolute quantification of target transcript in copies per input samples. As such, its application in validation of high throughput screening with Microarray (MA) or Next Generation Sequencing may provide a cost-effective practical solution. To validate the MA identified genomic biomarkers of appendicitis and bacterial respiratory infections, we have applied the ddPCR on whole blood RNA from patients with acute appendicitis and respiratory infections, as well as on control subjects enrolled in the GWU IRB approved study. The MA transcript profiling identified 37 differentially expressed genes (DEG) in appendicitis versus abdominal pain patients and several strong biomarkers of respiratory infections were discovered during data analysis. The DEG list contained three major ontologies: infection-related, inflammation-related, and ribosomal processing. The detected transcripts were validated using ddPCR on 70 patients and confirmed the patterns detected by MA or NGS alone. ddPCR may serve as a robust tool for validation of large scale genomic biomarkers discovery experiments and could be implemented into clinical practice once the biomarkers are validated
