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A Robust Protocol to Quantify Circulating Cancer Biomarker MicroRNAs

  • Emma Bell
  • Hannah L. Watson
  • Shivani Bailey
  • Matthew J. Murray
  • Nicholas Coleman
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1580)

Abstract

Reverse-transcriptase quantitative PCR (RT-qPCR) is a widely used method for quantifying microRNAs (miRNAs) in cells and tissues. However, the quantification of miRNAs in the circulation presents specific challenges. Here, we describe an optimized protocol using a small amount of input material to assess serum sample quality and quantify levels of a panel of up to 20 miRNAs. This is achieved by multiplexing Taqman miRNA stem-loop primers in the reverse transcription step. An additional multiplexed pre-amplification step is used to increase the sensitivity of the final quantification step, which is carried out using standard Taqman qPCR methodology.

Key words

MicroRNA Serum Plasma Cerebrospinal fluid Pre-amplification RT-qPCR 

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of PathologyUniversity of Cambridge, CambridgeCambridgeUK
  2. 2.Department of Paediatrics, Haematology and OncologyAddenbrooke’s HospitalCambridgeUK
  3. 3.Department of PaediatricsUniversity of Cambridge, Addenbrooke’s HospitalCambridgeUK
  4. 4.Department of HistopathologyAddenbrooke’s HospitalCambridgeUK
  5. 5.AstraZenecaCambridge Science ParkCambridgeUK

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