Measuring expression levels of small regulatory RNA molecules from body fluids and formalin-fixed, paraffin-embedded samples

Adrienn Gyongyosi, Otto Docs, Zsolt Czimmerer, Laszlo Orosz, Attila Horvath, Olga Török, Gabor Mehes, Laszlo Nagy, Balint L. Balint

Research output: Chapter in Book/Report/Conference proceedingChapter

4 Citations (Scopus)

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.

Original languageEnglish
Title of host publicationRNA Mapping
Subtitle of host publicationMethods and Protocols
PublisherHumana Press Inc.
Pages105-119
Number of pages15
ISBN (Print)9781493910618
DOIs
Publication statusPublished - 2014

Publication series

NameMethods in Molecular Biology
Volume1182
ISSN (Print)1064-3745

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Keywords

  • Cancer
  • FFPE
  • RT-qPCR
  • Stem-loop
  • Urine
  • miRNA

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Gyongyosi, A., Docs, O., Czimmerer, Z., Orosz, L., Horvath, A., Török, O., Mehes, G., Nagy, L., & Balint, B. L. (2014). Measuring expression levels of small regulatory RNA molecules from body fluids and formalin-fixed, paraffin-embedded samples. In RNA Mapping: Methods and Protocols (pp. 105-119). (Methods in Molecular Biology; Vol. 1182). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-1062-5_10