title: Measuring expression levels of small regulatory RNA molecules from body fluids and formalin-fixed, paraffin-embedded samples.
abstract: 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 the 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.
authors: Department of Biochemistry and Molecular Biology, Center for Clinical Genomics and Personalized Medicine, University of Debrecen Medical and Health Science Center (UD MHSC), Nagyerdei krt. 98, Debrecen, Hungary.
authors: Adrienn
authors: A
authors: Gyongyosi
authors: Otto
authors: O
authors: Docs
authors: Zsolt
authors: Z
authors: Czimmerer
authors: Laszlo
authors: L
authors: Orosz
authors: Attila
authors: A
authors: Horvath
authors: Olga
authors: O
authors: Török
authors: Gabor
authors: G
authors: Mehes
authors: Laszlo
authors: L
authors: Nagy
authors: Balint L
authors: BL
authors: Balint
mesh_terms: D000818:Animals; D001826:Body Fluids / Q000737:chemistry*; D020869:Gene Expression Profiling / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D035683:MicroRNAs / Q000032:analysis; D010232:Paraffin; D016612:Paraffin Embedding; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: C576078:MIRN325 microRNA, human; C585821:MIRN515 microRNA, human; D035683:MicroRNAs; D010232:Paraffin