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Multiplex Analyses Using Real-Time Quantitative PCR

  • Steve F. C. Hawkins
  • Paul C. Guest
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1546)

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.

Key words

qPCR Fluorescent dyes Taq polymerase Quantitation mRNA cDNA Amplicon Multiplex analysis 

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Bioline Reagents Limited, Unit 16The Edge Business CentreLondonUK
  2. 2.Laboratory of Neuroproteomics, Department of Biochemistry and Tissue Biology, Institute of BiologyUniversity of Campinas (UNICAMP)CampinasBrazil

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