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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...
Reporter Genes02:11

Reporter Genes

Reporter genes are a type of protein-coding gene that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually produce visually identifiable characteristics like fluorescence and luminescence when expressed along with the gene of interest. Thus, reporter genes “report” the presence or absence of genes of interest in an organism, determine the gene expression pattern, or track the physical location of a DNA segment or protein in the cell.
Commonly used reporter...

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Related Experiment Video

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Genotyping of Plant and Animal Samples without Prior DNA Purification
11:00

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Published on: September 24, 2012

RT-PCR heteroduplex analysis permits differentiation of transgene and host gene expression in a transgenic animal

W Duan1, H Ding, W G Zhu

  • 1The Ohio State University College of Medicine and Public Health, Columbus 43210-1240, USA.

Biotechniques
|July 26, 2002
PubMed
Summary

This study introduces RT-PCR heteroduplex analysis, a novel method to distinguish transgene expression from homologous wild-type genes in animal models. This technique is effective even with highly conserved DNA sequences, simplifying gene expression analysis.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Evaluating transgene expression in animal models is challenging due to sequence conservation with host genes.
  • Gene silencing can further complicate the accurate assessment of transgene and endogenous gene expression.

Purpose of the Study:

  • To develop and validate a method for differentiating transgene expression from homologous wild-type gene expression.
  • To address challenges posed by highly conserved DNA sequences between transgenes and host genes.

Main Methods:

  • Reverse Transcription Polymerase Chain Reaction (RT-PCR) heteroduplex analysis was employed.
  • Primers were designed to amplify conserved regions of the p53 gene (DNA binding domain) from human and mouse mRNA.
  • Analysis was performed on samples from transgenic and wild-type mice.

Main Results:

  • RT-PCR heteroduplex analysis successfully differentiated transgene expression from wild-type gene expression in all tested samples.
  • Heteroduplex bands were consistently observed in transgenic samples and absent in wild-type controls.
  • The method identified a silenced transgene in one sample without sequencing or labeling.

Conclusions:

  • RT-PCR heteroduplex analysis is an effective, user-friendly method for screening numerous samples rapidly.
  • This technique is particularly valuable for detecting transgene expression when species-specific primers cannot be designed due to high sequence homology.