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Persistent DNA contamination in competitive RT-PCR using cRNA internal standards: identity, quantity, and control
Jennifer L K Matthews1, May Chung, Robert John Matyas
1University of Calgary, Faculty of Medicine, Alberta, Canada.
Biotechniques
|June 21, 2002
Summary
Accurate mRNA quantification using competitive RT-PCR requires strict cRNA quality control. DNA contamination in cRNA standards, often overlooked, leads to overestimated mRNA copy numbers and requires correction.
Area of Science:
- Molecular Biology
- Biochemistry
Background:
- Accurate mRNA quantification via competitive RT-PCR (reverse transcription polymerase chain reaction) is crucial.
- Internal cRNA standards must be free of DNA contamination for reliable results.
Purpose of the Study:
- To evaluate methods for removing template DNA from cRNA internal standards.
- To develop a strategy for quantifying DNA contamination in cRNA standards.
Main Methods:
- Assessed the efficacy of RNase-free DNase I in removing template DNA.
- Investigated DNA reduction, selective extraction, and gel fractionation.
- Developed an "inverted" competitive RT-PCR method to quantify DNA contamination.
Main Results:
- RNase-free DNase I is insufficient and can degrade cRNA.
- Standard DNA removal techniques failed to eliminate contamination completely.
- A small but significant percentage of DNA contamination consistently remained in in vitro transcribed cRNA.
Conclusions:
- Uncorrected DNA contamination in cRNA standards leads to systematic overestimation of mRNA.
- The "inverted" competitive RT-PCR method effectively quantifies DNA contamination.
- Correction for DNA contamination is essential for accurate mRNA quantification.