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Methods Mol Biol. 2017;1580:265-279. doi: 10.1007/978-1-4939-6866-4_18.

A Robust Protocol to Quantify Circulating Cancer Biomarker MicroRNAs.

Author information

1
Department of Pathology, University of Cambridge, Cambridge, Tennis Court Road, Cambridge, CB2 1QP, UK.
2
AstraZeneca, Cambridge Science Park, Cambridge, CB4 0FZ, UK.
3
Department of Pathology, University of Cambridge, Cambridge, Tennis Court Road, Cambridge, CB2 1QP, UK. mjm16@cam.ac.uk.
4
Department of Paediatrics, Haematology and Oncology, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 0QQ, UK. mjm16@cam.ac.uk.
5
Department of Paediatrics, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 0QQ, UK. mjm16@cam.ac.uk.
6
Department of Pathology, University of Cambridge, Cambridge, Tennis Court Road, Cambridge, CB2 1QP, UK. nc109@cam.ac.uk.
7
Department of Histopathology, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 0QQ, UK. nc109@cam.ac.uk.

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.

KEYWORDS:

Cerebrospinal fluid; MicroRNA; Plasma; Pre-amplification; RT-qPCR; Serum

PMID:
28439839
DOI:
10.1007/978-1-4939-6866-4_18
[Indexed for MEDLINE]

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