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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

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Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells
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Analysis of conditional gene deletion using probe based Real-Time PCR.

Britta Weis1, Joachim Schmidt, Frank Lyko

  • 1Department of Gastroenterology and Hepatology, University Hospital Freiburg, Hugstetter Str 49, 79106 Freiburg, Germany.

BMC Biotechnology
|October 19, 2010
PubMed
Summary

Real-Time PCR with a loxP site-directed probe accurately measures Cre-lox recombination efficiency. This sensitive method overcomes SYBR Green limitations for precise 1lox/2lox allele ratio analysis in mouse genetics.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Conditional gene deletion via Cre-lox recombination is crucial in mouse genetics.
  • Incomplete recombination leads to mixed 1lox (truncated) and 2lox (functional) alleles.
  • Conventional Southern Blotting for allele ratio analysis is laborious, DNA-intensive, and lacks sensitivity.

Purpose of the Study:

  • To evaluate Real-Time PCR for accurate quantification of 1lox/2lox allele ratios.
  • To develop a more specific and sensitive method than SYBR Green-based Real-Time PCR.
  • To establish a universally applicable assay for Cre-lox recombination efficiency.

Main Methods:

  • SYBR Green-based Real-Time PCR was initially assessed but showed erroneous melting curve peaks.
  • A probe-based Real-Time PCR method utilizing a universal probe targeting the loxP site was developed.
  • The method was validated in Fabpl4× at -132-Cre transgenic mice.

Main Results:

  • SYBR Green Real-Time PCR yielded unreliable data due to potential alternate hybridization products.
  • Probe-based Real-Time PCR accurately detected subtle differences in 1lox/2lox allele ratios.
  • The developed assay demonstrated high accuracy and sensitivity, consistent with published data.

Conclusions:

  • Real-Time PCR with a loxP site-directed probe offers a highly accurate and sensitive method for quantifying 1lox/2lox allele ratios.
  • The universal probe design allows for broad applicability across various Cre-lox mediated gene deletion models.
  • This assay provides a convenient and improved alternative to conventional methods for assessing recombination efficiency.