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Related Concept Videos

Real Time RT-PCR02:57

Real Time RT-PCR

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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...
62.7K

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Real-time quantitative PCR: A tool for absolute and relative quantification.

Ravikumar Harshitha1, Duraipandian Rex Arunraj1

  • 1Department of Genetic Engineering, SRM Institute of Science and Technology, Kattankulathur, India.

Biochemistry and Molecular Biology Education : a Bimonthly Publication of the International Union of Biochemistry and Molecular Biology
|June 16, 2021
PubMed
Summary

This study details real-time quantitative PCR (RT-qPCR), covering absolute and relative quantification methods for diverse applications. Laboratory experiments explore key principles like primer design and PCR efficiency for accurate gene expression analysis.

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PCR efficiencyabsolute and relative quantificationcopy numberfold changequantitative real time PCR

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Genomics

Background:

  • Real-time quantitative PCR (RT-qPCR) monitors PCR amplification in real time.
  • RT-qPCR is essential for accurate quantification in various scientific disciplines.
  • Understanding RT-qPCR principles is crucial for reliable experimental outcomes.

Purpose of the Study:

  • To provide a comprehensive laboratory guide to RT-qPCR principles and data analysis.
  • To differentiate between absolute and relative quantification methods.
  • To offer practical insights into primer characteristics, PCR efficiency, and melt curve analysis.

Main Methods:

  • Laboratory experiments designed to illustrate RT-qPCR fundamentals.
  • Manual and software-based data analysis techniques explored.
  • Focus on practical aspects of PCR optimization and validation.

Main Results:

  • Demonstration of absolute quantification for microbiological and biotechnological applications.
  • Illustration of relative quantification for gene expression analysis in genomics.
  • Experimental validation of primer design, PCR efficiency, and melt curve analysis.

Conclusions:

  • This laboratory work equips students with essential RT-qPCR skills for undergraduate and graduate research.
  • Provides a foundational understanding for applying RT-qPCR in diverse biological contexts.
  • Emphasizes the importance of optimizing PCR parameters for accurate quantification.