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.
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Acknowledgements
Balint L. Balint is a Szodoray fellow of the University of Debrecen Medical and Health Science Center, Hungary and is supported by the TÁMOP 4.2.4.A/2-11-1-2012-0001 grant implemented through the New Hungary Development Plan co-financed by the European Social Fund and the European Regional Development Fund. Jim Hesson of AcademicEnglishSolutions.com revised the English.
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Gyongyosi, A. et al. (2014). Measuring Expression Levels of Small Regulatory RNA Molecules from Body Fluids and Formalin-Fixed, Paraffin-Embedded Samples. In: Alvarez, M., Nourbakhsh, M. (eds) RNA Mapping. Methods in Molecular Biology, vol 1182. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1062-5_10
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DOI: https://doi.org/10.1007/978-1-4939-1062-5_10
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