A Circulating MicroRNA Panel for Malignant Germ Cell Tumor Diagnosis and Monitoring

Methods Mol Biol. 2021:2195:225-243. doi: 10.1007/978-1-0716-0860-9_15.

Abstract

Reverse transcription (RT) based quantitative PCR (qPCR) for quantifying microRNAs (miRNAs) in in the circulation presents specific challenges. Here, we describe an optimized research protocol to assess serum sample quality and quantify levels of a panel of four test miRNAs (miR-371a-3p, miR-372-3p, miR-373-3p, and miR-367-3p) that enables highly sensitive and specific malignant germ cell tumor (GCT) diagnosis and monitoring. This protocol utilizes a multiplex RT step using Taqman miRNA stem-loop primers. A multiplexed preamplification stage is then employed to increase the sensitivity of the final quantification step, which is performed using standard singleplex Taqman qPCR methodology.

Keywords: Circulating; Preamplification; RT-qPCR; Serum; microRNA.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biomarkers, Tumor*
  • Circulating MicroRNA*
  • Data Interpretation, Statistical
  • Humans
  • Multiplex Polymerase Chain Reaction
  • Neoplasms, Germ Cell and Embryonal / blood
  • Neoplasms, Germ Cell and Embryonal / diagnosis*
  • Neoplasms, Germ Cell and Embryonal / genetics*
  • Nucleic Acid Amplification Techniques
  • Prognosis
  • Quality Control
  • Real-Time Polymerase Chain Reaction / methods
  • Real-Time Polymerase Chain Reaction / standards
  • Workflow

Substances

  • Biomarkers, Tumor
  • Circulating MicroRNA