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Fluorescent Oligonucleotide Probes for the Quantification of RNA by Real-Time qPCR

  • Florent BusiEmail author
Protocol
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Part of the Methods in Molecular Biology book series (MIMB, volume 2113)

Abstract

Quantitative real-time PCR (qPCR) is a widely adopted technique used for scientific, clinical, diagnostic, or quality control purposes. One of the main applications of qPCR is gene expression analysis, although mutation detection, genotyping, DNA detection, and quantification (from pathogens or genetically modified organisms) are also investigated using this technique.

Although nonspecific detection based on DNA-binding dyes (including SYBR Green I) offers versatility in qPCR assays, detection of the PCR product using fluorescent probes confers higher specificity and sensitivity to assays, justifying the use of fluorescent probes as a detection method.

This chapter seeks to propose a procedure for the design of qPCR assays using fluorescent hydrolysis probe technology. Particular attention will be paid to explaining the steps necessary to ensure the specificity of the oligonucleotides used as primers or fluorescent probes.

Key words

qPCR Hydrolysis probes Fluorophore Quencher 

Abbreviations

bp

Base pair

BHQ(-1 or -2)

Black Hole Quencher (-1 or -2)

Cq

Quantification cycle

FAM

Carboxyfluorescein

gDNA

Genomic DNA; DABCYL: 4-4-dimethylamino-phenyl-azo-benzoic acid

HEX

6-Carboxy-2,4,4,5,7,7-hexachlorofluorescein

JOE

6-Carboxy4′,5′-dichloro-2′,7′-dimethoxyfluorescein

nt

Nucleotide

qPCR

Quantitative real-time PCR

ROX

Carboxy-X-rhodamine

RT

Reverse transcription reaction

ss/ds

Single/double stranded

TAMRA

Carboxytetramethylrhodamine

TET

6-Carboxy-2′,4,7,7′-tetrachlorofluorescein

Tm

Melting temperature

Notes

Acknowledgments

This work was supported by University Paris Diderot and CNRS.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Unité de Biologie Fonctionnelle et AdaptativeUniversité de Paris, CNRS UMR 8251ParisFrance

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