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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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Related Experiment Video

Updated: Jun 14, 2026

Simultaneous Quantification of T-Cell Receptor Excision Circles (TRECs) and K-Deleting Recombination Excision Circles (KRECs) by Real-time PCR
14:14

Simultaneous Quantification of T-Cell Receptor Excision Circles (TRECs) and K-Deleting Recombination Excision Circles (KRECs) by Real-time PCR

Published on: December 6, 2014

A standard curve based method for relative real time PCR data processing.

Alexey Larionov1, Andreas Krause, William Miller

  • 1Breast Unit, Western General Hospital, Edinburgh, UK. alexey_larionov@hotmail.com

BMC Bioinformatics
|March 23, 2005
PubMed
Summary
This summary is machine-generated.

This study presents a new standard curve method for processing real-time PCR data, offering a reliable and simple alternative to PCR efficiency calculations for gene expression analysis.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Genetics

Background:

  • Real-time PCR is a precise gene expression measurement tool.
  • Data processing significantly impacts real-time PCR results.
  • Current methods rely on standard curves or PCR efficiency assessment.

Purpose of the Study:

  • Implement and validate a standard curve method for relative real-time PCR.
  • Provide a reliable and simple alternative to PCR efficiency calculations.
  • Discuss advantages and limitations of the standard curve method.

Main Methods:

  • Developed a procedure for processing real-time PCR data using standard curves.
  • Filtered noise from raw fluorescence readings (smoothing, baseline subtraction, amplitude normalization).
  • Automatically selected optimal thresholds and derived crossing points (CPs).
  • Calculated means and variances for CPs and traced variances to final results.

Main Results:

  • The procedure minimizes operator involvement and provides statistical assessment of intra-assay variation.
  • Standard curve method is a reliable and simple alternative to PCR efficiency-based calculations.
  • Limitations of parametric statistical methods and amplitude normalization were analyzed and deemed suitable for routine practice.

Conclusions:

  • A validated standard curve-based procedure for PCR data processing was compiled.
  • Standard curve design is a reliable and simple alternative for relative real-time PCR.
  • The method is suitable for routine laboratory practice.