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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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Introductory Analysis and Validation of CUT&#38;RUN Sequencing Data
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Reverse transcription-quantitative polymerase chain reaction: description of a RIN-based algorithm for accurate data

Alexandre Ho-Pun-Cheung1, Caroline Bascoul-Mollevi, Eric Assenat

  • 1INSERM U, Val d'Aurelle Cancer Institute, Montpellier, France. alex.hpc@valdorel.fnclcc.fr

BMC Molecular Biology
|April 17, 2009
PubMed
Summary

Accurate gene expression requires accounting for RNA degradation. A new normalization method for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) corrects for RNA integrity loss, significantly reducing errors in human biopsy samples.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Genomics

Background:

  • Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a standard for mRNA quantification.
  • Accurate normalization is crucial for reliable RT-qPCR data.
  • Quantitating total RNA is a common normalization method but doesn't account for RNA integrity.

Purpose of the Study:

  • To evaluate the impact of RNA degradation on RT-qPCR gene expression measurements.
  • To develop an improved normalization method for RT-qPCR that corrects for RNA integrity loss.
  • To validate the new normalization strategy in human rectal cancer biopsies.

Main Methods:

  • Assessed the effect of RNA degradation on the expression of nine genes using RT-qPCR.
  • Developed a corrective algorithm to compensate for RNA integrity loss during normalization.
  • Applied the improved normalization strategy to quantify gene expression in rectal cancer biopsies.

Main Results:

  • RNA degradation can introduce up to 100% error in gene expression measurements when normalizing to total RNA.
  • The novel normalization method reduced the average error in quantitative measurements to 8%.
  • The strategy enabled more reliable gene expression comparisons in human rectal cancer samples.

Conclusions:

  • A new normalization method for RT-qPCR data has been developed.
  • This method compensates for RNA integrity loss, enabling accurate gene expression quantification.
  • The approach is particularly valuable for analyzing degraded human biopsy samples.