Improved urine samples collection for DNA/RNA purification and its application in bladder cancer detection.

Abstract

e18199 Background: Urine represents an extremely important body fluid for the sensitive and non-invasive detections of multiple diseases including urinary tract infections and cancers. Recently, urine nucleic acids based biomarkers has gained popularity as the preferred diagnostic methods. However, DNA and RNA in urine are prone to degradation and conventional urine storage (i.e. sample freezing and the addition of additives) have limited positive impacts on the nucleic acids quality. Therefore, to ensure the robust urinalysis, a well-standardized procedure of urine sample collection, storage, and nucleic acid purification should be developed and streamlined. Methods: We developed a novel technology, Urine Conditioning Buffer (UCB) which effectively preserves total DNA and RNA in urine samples for at least 1 month at ambient temperatures, abating the high costs of cold chain. Importantly, UCB inhibits microbial growth, reducing the risk of biohazard exposure. Additionally, the combination of UCB and Quick-DNA Urine Kit (Zymo Research) allows the isolation of either cellular, cell-free, or total DNA from urine samples. Results: We further validated our technology by conducting a pre-clinical study. The DNA methylation level of the 13 unique bladder cancer biomarkers (developed at Zymo Research) was analyzed in a cohort of 53 individuals (33 bladder cancer patients and 20 healthy individuals). Collected urine samples were preserved in UCB and total urine DNA was isolated using Quick-DNA Urine Kit. Urine DNA was analyzed in parallel using two different approaches: a bisulfite conversion based assay and a non-bisulfite based assay. Despite the methodology differences, both assays perfectly discriminated bladder cancer from control samples. This study indicates that UCBpreserves the urine DNA integrity, a prerequisite for the successful urine DNA and RNA based testing. Conclusions: Here, we present a successful application of our unique technology in bladder cancer diagnosis, which is the 5th most common cancer in US. Furthermore, it demonstrates that we have developed a total solution for urine based molecular testing covering sample collection, stabilization, purification, and detection platform.