Targeting the human urine RNAome for tumor diagnostics by qPCR

Abstract
In the last decade urine nucleic acids (DNA, RNA) and their epigenetic modifications (DNA methylations) have been established as a valuable diagnostic ressource for a variety of human diseases, especially of the urogenital system (bladder, kidney, prostate). Of particular interest are tumor marker RNA molecules potentially reflecting the – by disease progression – altered in situ cellular expression pattern in the urinary fluid. Hence, an advanced multi-marker analysis by qPCR could allow a comprehensive non-invasive characterization of the organically caused disorders. RNA is released into the urine compartment by several physiological mechanisms including natural cell shedding, active secretion, cell lysis, apoptosis and necrosis. The total urine RNA content (urine RNAome: mRNA, miRNA, ncRNA, other small RNAs) consists of a cell-associated (ucRNA; urine cellular RNA) and a cell-free (ufRNA; urine free RNA) component. We present a brief overview of molecular urine RNA-based diagnostics with a focus on prostate cancer. Urine constitutes a chemically harsh environment for RNA integrity and the development of a standardized preanalytical regime will determine the future success of the field. We were able to quantitate selected prostate cancer biomarker mRNAs/miRNAs by LightCycler® 480 Real-Time PCR System qPCR1. We found that the major obstacle to a meaningful gene expression profiling in prostate cancer urine (ucRNA and ufRNA) by qPCR is the dilution of the specific prostate cancer cell-derived RNA signals in a non-specific background (consisting of non-malignant prostate and non-prostate cells). Theoretically four methods could be devised to circumvent the tumor RNA thinning effect: sophisticated differential hybridization methods, single cell qPCR of separated cancer cells, immune magnetic cancer cell enrichment and urinary cancer cell tissue culture prior to the genetic analysis. All mentioned techniques have advantages and limitations for the development of clinically useful quantitative gene expression assays. For instance, ucRNA-based tests depend on tumor marker mRNA expression stability in the presence of urine for at least two hours (bladder resting time after a prostate massage). Therefore one of our present research goals is to measure biomarker mRNA expression after urine exposure for different time points in a prostate cancer cell culture model by qPCR. In summary, biostatistics-supported RNA quantification in urine is on its way to revolutionize the field of kidney, bladder and prostate cancer molecular diagnostics (supported by Austrian National Bank P11491, Milestone Dr. Erwin Pröll Future Prize for Innovation, RIZ Genius Award). 1Schöller, A., Mayer, M., Cionca, S., Grubmüller, K., Wang, M., Hu, Y., Stearns, M., and C. Freibauer (2009) Relative real time qPCR analysis of molecular urine markers (mRNA, miRNA) for prostate cancer. 3. Forschungsforum der österreichischen Fachhochschulen, 28 – 34, ISBN 978-853912850.