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Simple Summary Non-invasive tumor diagnosis includes liquid biopsy withdrawn from extracellular and extratissue samples including human urine in which RNA-based markers can be measured by various means.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.

[1]  Maoshan Chen,et al.  Liquid biopsy: an evolving paradigm for the biological characterisation of plasma cell disorders , 2021, Leukemia.

[2]  D. de Jong,et al.  Extracellular vesicle miRNA predict FDG‐PET status in patients with classical Hodgkin Lymphoma , 2021, Journal of extracellular vesicles.

[3]  T. Crnogorac-Jurcevic,et al.  Non-Invasive Biomarkers for Earlier Detection of Pancreatic Cancer—A Comprehensive Review , 2021, Cancers.

[4]  I. Endo,et al.  Urine as a Source of Liquid Biopsy for Cancer , 2021, Cancers.

[5]  G. Sczakiel,et al.  Transcriptome analyses of urine RNA reveal tumor markers for human bladder cancer: validated amplicons for RT-qPCR-based detection , 2021, Oncotarget.

[6]  F. Farinati,et al.  Liquid Biopsy in Hepatocellular Carcinoma: Where Are We Now? , 2021, Cancers.

[7]  M. Lenassi,et al.  Extracellular Vesicles: A Novel Tool Facilitating Personalized Medicine and Pharmacogenomics in Oncology , 2021, Frontiers in Pharmacology.

[8]  Jiayin Hou,et al.  Combination of Urine Exosomal mRNAs and lncRNAs as Novel Diagnostic Biomarkers for Bladder Cancer , 2021, Frontiers in Oncology.

[9]  Shizhong Xu,et al.  A urine-based DNA methylation assay to facilitate early detection and risk stratification of bladder cancer , 2021, Clinical epigenetics.

[10]  V. Constâncio,et al.  Urinary Extracellular Vesicles as Potential Biomarkers for Urologic Cancers: An Overview of Current Methods and Advances , 2021, Cancers.

[11]  S. Sleijfer,et al.  Detection of tumor-derived extracellular vesicles in plasma from patients with solid cancer , 2020, BMC cancer.

[12]  F. Skorpen,et al.  DNA methylation markers detected in blood, stool, urine, and tissue in colorectal cancer: a systematic review of paired samples , 2020, International Journal of Colorectal Disease.

[13]  Ronnie H. Fang,et al.  Natural display of nuclear-encoded RNA on the cell surface and its impact on cell interaction , 2020, Genome Biology.

[14]  G. Mias,et al.  Characterizing Extracellular Vesicles and Their Diverse RNA Contents , 2020, Frontiers in Genetics.

[15]  Andrew J. Lindsay,et al.  A comparative study of extracellular vesicle-associated and cell-free DNA and RNA for HPV detection in oropharyngeal squamous cell carcinoma , 2020, Scientific Reports.

[16]  W. Cho,et al.  The potential of circulating exosomal RNA biomarkers in cancer , 2020, Expert review of molecular diagnostics.

[17]  G. Sczakiel,et al.  Oncoprotein 18 is necessary for malignant cell proliferation in bladder cancer cells and serves as a G3-specific non-invasive diagnostic marker candidate in urinary RNA , 2020, bioRxiv.

[18]  Jun-Pil Jee,et al.  Extracellular Vesicles: The Next Frontier in Regenerative Medicine and Drug Delivery. , 2020, Advances in experimental medicine and biology.

[19]  A. Hendrix,et al.  Targets, pitfalls and reference materials for liquid biopsy tests in cancer diagnostics. , 2019, Molecular aspects of medicine.

[20]  C. Ritch,et al.  Urinary biomarkers in bladder cancer: where do we stand? , 2019, Current opinion in urology.

[21]  S. Mowla,et al.  Urinary exosomal expression of long non-coding RNAs as diagnostic marker in bladder cancer , 2018, Cancer management and research.

[22]  T. Todenhöfer,et al.  Can urinary biomarkers replace cystoscopy? , 2018, World Journal of Urology.

[23]  A. Carracedo,et al.  Vesicle-MaNiA: extracellular vesicles in liquid biopsy and cancer. , 2016, Current opinion in pharmacology.

[24]  P. Laktionov,et al.  Comparative Study of Extracellular Vesicles from the Urine of Healthy Individuals and Prostate Cancer Patients , 2016, PloS one.

[25]  Jason L. Townson,et al.  Prostate extracellular vesicles in patient plasma as a liquid biopsy platform for prostate cancer using nanoscale flow cytometry , 2016, Oncotarget.

[26]  P. Laktionov,et al.  Extracellular Nucleic Acids in Urine: Sources, Structure, Diagnostic Potential , 2015, Acta naturae.

[27]  Dieter Jocham,et al.  A robust methodology to study urine microRNA as tumor marker: microRNA-126 and microRNA-182 are related to urinary bladder cancer. , 2010, Urologic oncology.

[28]  D. Jocham,et al.  Detailed technical analysis of urine RNA-based tumor diagnostics reveals ETS2/urokinase plasminogen activator to be a novel marker for bladder cancer. , 2007, Clinical chemistry.

[29]  A. V. Starikov,et al.  Investigation of tumor-derived extracellular DNA in blood of cancer patients by methylation-specific PCR. , 2004 .

[30]  J. Warnecke,et al.  Improved Conditions for Isolation and Quantification of RNA in Urine Specimens , 2004, Annals of the New York Academy of Sciences.