MicroRNAs are involved in the regulation of various pathophysiological processes such as immune regulation and cancer. Next-generation sequencing methods enable us to monitor their presence in various types of samples but we need flexible methods for validating datasets generated by high throughput methods. Here we describe the detailed protocols to be used with our MiRNA Primer Design Tool assay design system. The presented methods allow the flexible design of the oligonucleotides needed for

more »

... e RT-qPCR detection of any variant of small regulatory RNA molecules from virtually any species. This method can be used to measure miRNA levels from formalin-fixed, paraffin-embedded (FFPE) samples and various body fluids. As an example, we show the results of the hsa-miR-515-3p, hsa-miR-325, and hsa-miR-155 quantification using a specific UPL probe (Universal Probe Library) and a stem-loop RT-qPCR assay. The small nucleolar RNA RNU43 is used as endogenous control for normalization of the results. Urine from healthy pregnant women and FFPE samples from patients diagnosed with colorectal cancer and treated with antibody-based anti-EGFR monotherapy were used as samples. DEOEC RKEB/IKEB 3092-2010). All patients were informed about the purpose of sample collection and signed an informed consent form. FFPE samples FFPE samples were obtained during routine diagnostic procedures for histological and molecular analysis performed in the Department of Pathology, University of Debrecen. All samples had KRAS mutant genotype confirmed by capillary sequencing and were divided into two groups of five patients according to disease-free survival after anti-EGFR therapy (good and poor responders). Samples were collected according to rules and regulations of the University of Debrecen, Medical and Health Science Center, with the approval of the local ethics committee (file number: RKEB/IKEB 3856-2013). Reagents and equipments