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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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Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards
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Effective Alu repeat based RT-Qpcr normalization in cancer cell perturbation experiments.

Ali Rihani1, Tom Van Maerken, Filip Pattyn

  • 1Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium.

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|August 27, 2013
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Alu repeats offer stable expression for normalizing gene levels in cancer cell experiments. This finding provides a reliable internal control for reverse transcription quantitative polymerase chain reaction (RT-qPCR) data in cancer research.

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Area of Science:

  • Molecular Biology
  • Cancer Research
  • Genomics

Background:

  • Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a standard method for measuring messenger RNA (mRNA) levels.
  • Accurate normalization of RT-qPCR data relies on selecting stable internal control reference genes.
  • Cancer cell manipulations can significantly alter gene expression, impacting the reliability of commonly used reference genes.

Purpose of the Study:

  • To evaluate the expression stability of 11 commonly used reference genes in various cancer cell lines.
  • To identify reliable internal control assays for normalizing RT-qPCR data in cancer cell perturbation experiments.

Main Methods:

  • Utilized the geNorm algorithm within the qbase+ software package to rank candidate reference genes.
  • Assessed the expression stability of 11 reference genes across 19 experiments involving diverse cancer cell lines and perturbations.

Main Results:

  • Most candidate reference genes exhibited variable expression stability under different experimental conditions.
  • Expressed Alu repeats demonstrated consistent expression stability across all perturbation experiments.
  • Alu repeats were ranked among the top reference assays with acceptable average expression stability values (M<0.5).

Conclusions:

  • Alu repeats are proposed as a reliable reference assay for normalizing RT-qPCR data in cancer cell perturbation experiments.
  • The stable expression of Alu repeats provides a robust internal control, enhancing the accuracy of gene expression studies in cancer research.