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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...
RACE - Rapid Amplification of cDNA Ends02:35

RACE - Rapid Amplification of cDNA Ends

Rapid Amplification of cDNA Ends, or RACE, is one of the most effective methods to obtain a full-length cDNA from an mRNA sequence between a known internal region to the unknown sequence at the 5’ or 3’ end. The unknown region is cloned in the cDNA by a gene-specific primer that binds the known end, and a hybrid primer that attaches a predefined anchor sequence to the unknown end of the cDNA. The sequence in between is amplified by PCR with an anchor primer and a gene-specific primer.
Since the...

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Related Experiment Video

Updated: Jul 7, 2026

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

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Published on: March 30, 2015

Improved normalization of real-time reverse transcriptase polymerase chain reaction data using an external RNA

Stian Ellefsen1, Kåre-Olav Stensløkken, Guro K Sandvik

  • 1Physiology Programme, Department of Molecular Biosciences, University of Oslo, 0316 Oslo, Norway. stian.ellefsen@hil.no

Analytical Biochemistry
|February 26, 2008
PubMed
Summary

A novel external RNA control method improves real-time RT PCR data normalization. This approach accurately quanties gene expression, especially under physiological stress, overcoming limitations of current methods.

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Area of Science:

  • Molecular Biology
  • Gene Expression Analysis
  • Physiological Stress Response

Background:

  • Real-time RT PCR (reverse transcriptase polymerase chain reaction) data normalization lacks a validated standard.
  • Current normalization methods are often inadequate, especially during physiological challenges.
  • Accurate gene expression quantification is crucial for understanding biological responses.

Purpose of the Study:

  • To develop and validate a novel external RNA control approach for real-time RT PCR.
  • To assess the accuracy and necessity of this method in experiments with severe physiological challenges.
  • To improve the reliability of gene expression data normalization.

Main Methods:

  • Developed an external RNA control method adding RNA to tissue on a per unit weight basis.
  • Applied the method to analyze reference gene (beta-actin, cyclophilin A, glyceraldehyde 3-phosphate dehydrogenase) expression in crucian carp brain and heart under normoxic and anoxic conditions.
  • Compared normalization results with traditional methods and assessed target gene (HSC70) expression.

Main Results:

  • Internal RNA control genes showed significant expression differences between experimental groups, particularly in the heart.
  • The external RNA control approach provided more accurate normalization of target genes.
  • A 2.5-fold increase in stress-response gene HSC70 expression was detected, which was missed by beta-actin or geNorm normalization.

Conclusions:

  • The developed external RNA control approach significantly improves real-time RT PCR data normalization.
  • This method is accurate, suitable, and necessary for studies involving physiological challenges.
  • Standardized use of external RNA controls is recommended for reliable gene expression analysis.