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Open Access Highly Accessed Methodology article

A new reverse transcription-polymerase chain reaction method for accurate quantification

Yih-Horng Shiao

Author Affiliations

Laboratory of Comparative Carcinogenesis, Building 538, Room 205, National Cancer Institute at Frederick, National Institutes of Health, Frederick, MD 21702, USA

BMC Biotechnology 2003, 3:22  doi:10.1186/1472-6750-3-22

Published: 9 December 2003

Abstract

Background

Reverse transcription-polymerase chain reaction (RT-PCR) is a very sensitive technique to measure and to compare mRNA levels among samples. However, it is extremely difficult to maintain linearity across the entire procedure, especially at the step of PCR amplification. Specific genes have been used as baseline controls to be co-amplified with target genes to normalize the amplification efficiency, but development or selection of reliable controls itself has created a new challenge.

Results

Here, we describe a new quantitative RT-PCR to compare two mRNA samples directly without the requirement of synthetic control DNAs for reference. First, chimeric RT primers carrying gene-specific and universal PCR priming sequences with or without a linker for size distinction were utilized to generate cDNAs. The size-different cDNAs were then combined in a single reaction for PCR amplification using the same primer set. The two amplified products were resolved and detected with gel electrophoresis and fluorescence imaging. Relative abundance of the two products was obtained after a baseline correction.

Conclusion

This methodology is simple and accurate as indicated by equal amplification efficiency throughout PCR cycling. It is also easily implemented for many existing protocols. In addition, parameters affecting RT linearity are characterized in this report.