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[Reverse transcriptase PCR (RT-PCR) and quantitative-competitive PCR (QC-PCR)]
1Department of Obstetrics and Gynecology, College of Medicine, Ewha Womans University, Seoul, Korea.
Experimental & Molecular Medicine
|November 16, 2001
Summary
Quantitative PCR (qPCR) offers sensitive mRNA detection but lacks reliability. This study introduces competitive qPCR (QC-PCR) using an internal standard for accurate RNA and DNA quantitation, even with limited samples.
Area of Science:
- Molecular Biology
- Biochemistry
Context:
- Polymerase Chain Reaction (PCR) is highly sensitive for amplifying low-abundance mRNA but is unreliable for quantitation due to exponential amplification.
- Existing methods like Northern blots require large RNA amounts (≥10mg) and offer only semiquantitation.
Purpose:
- To develop a reliable and accurate method for mRNA and DNA quantitation using limited biological samples.
- To overcome the inherent variability and inaccuracy issues in standard PCR-based quantitation.
Summary:
- A competitive quantitative PCR (QC-PCR) method was developed using an internal standard with a deleted fragment of the target cDNA.
- This internal standard is coamplified with the target using identical primers and conditions, allowing for precise quantification.
- QC-PCR requires significantly less total RNA (1mg) compared to Northern blots and avoids discrepancies from variations in reverse transcription (RT) reaction kinetics.
Impact:
- QC-PCR provides a simple, inexpensive, and highly accurate method for quantifying specific target cDNA, mRNA, and DNA.
- Enables reliable gene expression analysis and molecular quantitation from small cell populations or limited tissue samples.
- Improves the accuracy of quantitative PCR by mitigating tube-to-tube and sample-to-sample variations.
