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Exclusive amplification of cDNA template (EXACT) RT-PCR to avoid amplifying contaminating genomic pseudogenes
R D Smith1, C W Ogden, M A Penny
1Imperial College of Science, Technology and Medicine, London, UK. daron.smith@ic.ac.uk
Biotechniques
|October 30, 2001
Summary
This study introduces a novel reverse transcription polymerase chain reaction (RT-PCR) method to prevent DNA contamination. The technique modifies complementary DNA (cDNA) to specifically exclude amplification from genomic DNA, improving RNA analysis accuracy.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Genomic DNA contamination in RNA samples complicates RT-PCR analysis, especially with pseudogenes.
- Existing DNA removal methods risk RNA degradation or loss, critical for low-expression targets.
Purpose of the Study:
- To develop a general RT-PCR technique to eliminate genomic DNA amplification without compromising RNA integrity.
- To enhance the accuracy of RT-PCR for gene expression studies by excluding DNA-based artifacts.
Main Methods:
- A novel sequence is ligated to the 5' tail of reverse-transcribed cDNA.
- This unique sequence serves as the annealing site for the reverse PCR primer.
- Genomic DNA lacks this specific sequence, preventing its amplification.
Main Results:
- The described method effectively prevents amplification from contaminating genomic DNA in RNA samples.
- This technique maintains RNA quantity and quality, crucial for sensitive gene expression analysis.
- It offers a reliable approach for distinguishing true cDNA amplicons from genomic DNA.
Conclusions:
- This innovative RT-PCR strategy provides a robust solution for mitigating DNA contamination.
- It enhances the reliability of gene expression studies by ensuring accurate amplification from cDNA.
- The method is broadly applicable to various RNA samples, particularly those with limited quantity or low-expressed transcripts.