Multiplex Analyses Using Real-Time Quantitative PCR

Steve F C Hawkins; Paul C Guest

doi:10.1007/978-1-4939-6730-8_8

Multiplex Analyses Using Real-Time Quantitative PCR

Methods Mol Biol. 2017:1546:125-133. doi: 10.1007/978-1-4939-6730-8_8.

Authors

Steve F C Hawkins¹, Paul C Guest²

Affiliations

¹ Bioline Reagents Limited, Unit 16, The Edge Business Centre, Humber Road, London, NW2 6EW, UK. steve.hawkins@bioline.com.
² Laboratory of Neuroproteomics, Department of Biochemistry and Tissue Biology, Institute of Biology, University of Campinas (UNICAMP), Campinas, Brazil.

PMID: 27896761
DOI: 10.1007/978-1-4939-6730-8_8

Abstract

Quantitative polymerase chain reaction (qPCR) is a routinely used method for the detection and quantitation of gene expression in real time. Multiplex qPCR requires the use of probe-based assays, in which each probe is labeled with a unique fluorescent dye, resulting in different observed colors for each assay. The signal from each dye is used to quantitate the amount of each target separately in the same tube or well. The availability to multiplex therefore allows the measurement of the expression levels of several targets or genes of interest quickly. Here, we describe a method using the SensiFAST and SensiFAST One-Step probe kits which allows simultaneous real-time quantitation of up to 5 amplicons.

Keywords: Amplicon; Fluorescent dyes; Multiplex analysis; Quantitation; Taq polymerase; cDNA; mRNA; qPCR.

Publication types

Review

MeSH terms

Biomarkers*
Humans
Molecular Diagnostic Techniques / methods
Molecular Diagnostic Techniques / standards
Multiplex Polymerase Chain Reaction* / methods
Multiplex Polymerase Chain Reaction* / standards
Real-Time Polymerase Chain Reaction* / methods
Real-Time Polymerase Chain Reaction* / standards
Reproducibility of Results
Sensitivity and Specificity

Substances

Biomarkers