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Related Concept Videos

Real Time RT-PCR02:57

Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...

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Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control
08:37

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

Published on: March 30, 2015

Performing quantitative reverse-transcribed polymerase chain reaction experiments.

Georges Lutfalla1, Gilles Uze

  • 1UMR5124, cc86 CNRS/Université Montpellier II, Montpellier, Cedex 05, France.

Methods in Enzymology
|August 30, 2006
PubMed
Summary

Real-time quantitative polymerase chain reaction (PCR) enables DNA quantification using fluorescent probes. This chapter details SYBR Green dye for reverse transcription PCR (RT-PCR) and discusses optimizing specificity, efficiency, and reproducibility for accurate results.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Quantitative polymerase chain reaction (PCR) has evolved significantly with technological advancements.
  • Real-time PCR instruments allow for continuous monitoring of DNA amplification.
  • Fluorescent probes are integral to quantifying amplified DNA in real-time PCR.

Purpose of the Study:

  • To focus on SYBR Green dye for quantifying specific cDNAs in reverse transcription PCR (RT-PCR) reactions.
  • To propose optimal reaction conditions for various real-time PCR instruments.
  • To discuss critical parameters for experimental setup, including specificity, efficiency, and reproducibility.

Main Methods:

  • Utilizing SYBR Green as a fluorescent label for DNA quantification.
  • Implementing real-time PCR instrumentation for on-line monitoring.
  • Performing reverse transcription followed by quantitative PCR (RT-PCR).

Main Results:

  • Established optimal reaction conditions for SYBR Green-based RT-PCR across different instruments.
  • Identified key parameters influencing the specificity, efficiency, and reproducibility of quantitative PCR assays.
  • Provided insights into the challenges and methodologies of relative quantification in RT-PCR.

Conclusions:

  • SYBR Green is an effective tool for quantitative RT-PCR when experiments are carefully designed and optimized.
  • Understanding and controlling parameters like specificity and efficiency are crucial for reliable DNA quantification.
  • This work facilitates accurate gene expression analysis using real-time quantitative PCR.