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

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RNA Spectroscopy

Part of the book series: Methods in Molecular Biology ((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.

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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

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Acknowledgments

This work was supported by University Paris Diderot and CNRS.

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Authors and Affiliations

  1. Unité de Biologie Fonctionnelle et Adaptative, Université de Paris, CNRS UMR 8251, Paris, France

    Florent Busi

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  1. Florent Busi
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Correspondence to Florent Busi .

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Editors and Affiliations

  1. Université de Paris, Paris, France, Laboratoire Léon Brillouin LLB, CEA, CNRS UMR12, Université Paris Saclay, Gif-sur-Yvette, France

    Véronique Arluison

  2. Synchrotron SOLEIL L’Orme des Merisiers Saint Aubin, Gif-sur-Yvette, France

    Frank Wien

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Busi, F. (2020). Fluorescent Oligonucleotide Probes for the Quantification of RNA by Real-Time qPCR. In: Arluison, V., Wien, F. (eds) RNA Spectroscopy. Methods in Molecular Biology, vol 2113. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0278-2_18

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  • DOI: https://doi.org/10.1007/978-1-0716-0278-2_18

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0277-5

  • Online ISBN: 978-1-0716-0278-2

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