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.
*Joint first authors.
† Joint senior authors.
†Joint senior authors.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Witwer KW (2015) Circulating microRNA biomarker studies: pitfalls and potential solutions. Clin Chem 61:56–63
Murray MJ et al (2016) A pipeline to quantify serum and cerebrospinal fluid microRNAs for diagnosis and detection of relapse in paediatric malignant germ-cell tumours. Br J Cancer 114:151–162
Blondal T et al (2013) Assessing sample and miRNA profile quality in serum and plasma or other biofluids. Methods 59:S1–S6
Pritchard CC et al (2012) Blood cell origin of circulating microRNAs: a cautionary note for cancer biomarker studies. Cancer Prev Res 5:492–497
Kirschner MB et al (2011) Haemolysis during sample preparation alters microRNA content of plasma. PLoS One 6:e24145
McDonald JS et al (2011) Analysis of circulating microRNA: preanalytical and analytical challenges. Clin Chem 57:833–840
Kroh EM et al (2010) Analysis of circulating microRNA biomarkers in plasma and serum using quantitative reverse transcription-PCR (qRT-PCR). Methods 50:298–301
Murray MJ et al (2014) Serum levels of mature microRNAs in DICER1-mutated pleuropulmonary blastoma. Oncogene 3:e87
Murray MJ et al (2015) Solid tumors of childhood display specific serum microRNA profiles. Cancer Epidemiol Biomarkers Prev 24:350–360
Mitchell PS et al (2008) Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A 105:10513–10518
Chen C et al (2005) Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33:e179
Tang F et al (2006) 220-plex microRNA expression profile of a single cell. Nat Protoc 1:1154–1159
Lao K et al (2006) Multiplexing RT-PCR for the detection of multiple miRNA species in small samples. Biochem Biophys Res Commun 343:85–89
Murray MJ et al (2011) Identification of MicroRNAs from the miR-371~373 and miR-302 clusters as potential serum biomarkers of malignant germ cell tumors. Am J Clin Pathol 135:119–125
Dieckmann KP et al (2016) MicroRNA miR-371a-3p – A novel serum biomarker of testicular germ cell tumors: evidence for specificity from measurements in testicular vein blood and in neoplastic hydrocele fluid. Urol Int 97:76–83
Wulfken LM et al (2011) MicroRNAs in renal cell carcinoma: diagnostic implications of serum miR-1233 levels. PLoS One 6:e25787
Meng X et al (2015) Diagnostic and prognostic potential of serum miR-7, miR-16, miR-25, miR-93, miR-182, miR-376a and miR-429 in ovarian cancer patients. Br J Cancer 113:1358–1366
Marabita F et al (2016) Normalization of circulating microRNA expression data obtained by quantitative real-time RT-PCR. Brief Bioinform 17:204–212
Schwarzenbach H et al (2015) Data normalization strategies for MicroRNA quantification. Clin Chem 61:1333–1342
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Bell, E., Watson, H.L., Bailey, S., Murray, M.J., Coleman, N. (2017). A Robust Protocol to Quantify Circulating Cancer Biomarker MicroRNAs. In: Dalmay, T. (eds) MicroRNA Detection and Target Identification. Methods in Molecular Biology, vol 1580. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6866-4_18
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6866-4_18
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6864-0
Online ISBN: 978-1-4939-6866-4
eBook Packages: Springer Protocols