Sampling, Logistics, and Analytics of Urine for RT-qPCR-based Diagnostics

SIMPLE SUMMARY: Non-invasive tumor diagnosis includes liquid biopsy withdrawn from extracellular and extratissue samples including human urine in which RNA-based markers can be measured by various means. RNA markers include short-chain RNA such as microRNA and long-chain linear and circular RNA. This study describes all steps between sample acquisition, sample stabilization, shipping, and the quantitative determination of RNA-based biomarkers by RT-qPCR that are related to non-coding and coding polymerase II transcripts including mRNA. We aim to provide a novel and thorough easy-to-perform description of all technical and logistics steps of urine RNA-based diagnostics. ABSTRACT: Body fluids in the context of cancer diagnosis are the primary source of liquid biopsy, i.e., biomarker detection that includes blood and serum, urine, and saliva. RNA represents a particular class of biomarkers because it is thought to monitor the current status of gene expression in humans, in organs, and if present, also in tumors. In case of bladder cancer, we developed a scheme that describes, in detail, all steps from the collection of urine samples from patients, stabilization of samples, their transportation, storage, and marker analysis by qPCR-based technology. We find that urine samples prepared according to this protocol show stability of RNA over more than 10 days at unchilled temperatures during shipping. A specific procedure of primer design and amplicon evaluation allows a specific assignment of PCR products to human genomics and transcriptomics data collections. In summary, we describe a technical option for the robust acquisition of urine samples and the quantitative detection of RNA-based tumor markers in case of bladder cancer patients. This protocol is for general use, and we describe that it works for any RNA-based tumor marker in urine of cancer patients.