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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...
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Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer Rate
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Quantitative rt-PCR.

P D Siebert1

  • 1Clontech Laboratories, Palo Alto, CA.

Methods in Molecular Medicine
|February 23, 2011
PubMed
Summary
This summary is machine-generated.

Quantitative analysis using reverse transcriptase polymerase chain reaction (RT-PCR) for mRNA is challenging due to its two enzymatic steps. However, with specific adaptations, RT-PCR can provide accurate quantitative mRNA measurements.

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Area of Science:

  • Molecular Biology
  • Biochemistry

Background:

  • Reverse transcriptase polymerase chain reaction (RT-PCR) is a highly sensitive technique for mRNA analysis.
  • Quantitative analysis using RT-PCR presents significant challenges.
  • The difficulty stems from the two sequential enzymatic steps: complementary DNA synthesis and polymerase chain reaction (PCR).

Purpose of the Study:

  • To address the challenges in obtaining quantitative data from RT-PCR.
  • To explore adaptations for accurate quantitative mRNA analysis using RT-PCR.

Main Methods:

  • Analysis of the two enzymatic steps in RT-PCR: complementary DNA synthesis and PCR amplification.
  • Evaluation of practical aspects and the inherent exponential nature of PCR.
  • Implementation of specific adaptations to the RT-PCR protocol.

Main Results:

  • The two sequential enzymatic steps, particularly the exponential nature of PCR, are primary obstacles to quantitative accuracy.
  • Practical considerations in performing RT-PCR also hinder precise quantification.
  • Specific adaptations can overcome these limitations to achieve accurate quantitative results.

Conclusions:

  • While inherently challenging, quantitative RT-PCR is achievable.
  • Adaptations to the RT-PCR methodology are crucial for reliable quantitative mRNA analysis.
  • RT-PCR can be a powerful tool for quantitative mRNA studies when properly implemented.