Cell-free microRNAs (miRNAs) have become one of the novel promising diagnostic and prognostic biomarkers for various diseases recently. Blood serum and plasma along with urine are the most common sources of clinically well, almost noninvasively available samples containing various types of miRNAs. Here, we present a protocol for a small-scale study investigating expression of several candidate miRNAs. Small-scale experiments may be worth investigating in cases where no information is available on miRNAs expression in particular diseases, for validation of previously published miRNAs with promising diagnostic potential, particularly in situations where follow-up study is aimed at validating miRNAs coming from array or NGS experiments, or where funding for these large-scale experiments is not available.
Using urine miRNAs expression as the novel diagnostic tools is challenging and currently this approach is still in its infancy. Therefore, various methods may result in different conclusions depending on clinical sample sets and differences among methods used for the miRNAs isolation and quantitation. In this protocol, we present the method evaluated in the study focused on cell-free urinary miRNAs in ovarian and endometrial cancers. We recommend using stabilization tubes for the urine collection, as this step may be necessary to stop activity of RNases. Further, routine real-time PCR methods are described. We demonstrate that assessment of urinary miRNAs expression may reveal as a feasible method to explore the potential for finding novel diagnostic and prognostic markers.
Urine MicroRNA Diagnostics Real-time PCR Ovarian cancer Endometrial cancer
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The financial support from the Charles University Prague (projects PRVOUK-P27/LF1/1, Progres Q28/LF1 and PRVOUK-P25/ LF1/2) and from the Ministry of Health of the Czech Republic (project FNBr 65269705) is appreciated. We would like to thank Avast Foundation, Ferona, a.s., ČEPS, a.s. and CEZ Foundation for their kind support of our research.
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