Rapid and Sensitive Closed-Tube Quantification of Human Interferon-γ mRNA by Reverse Transcription-PCR Utilizing Energy-Transfer Labeled Primers

Quantification of cytokine gene expression is essential for understanding the biological roles of cytokines in immune responses. Quantitative reverse transcription-PCR has become popular for the sensitive detection and quantification of mRNA (1). However, the necessity of post-PCR sample processing with the potential for carryover contamination of the amplicon makes it a difficult methodology for routine clinical testing. Using AmplifluorTM technology, we have developed a rapid and extremely sensitive assay for cytokine mRNA quantification. The high signal-to-noise ratio of the assay that is achieved by the unique structure of Amplifluor primers allows for closed-tube fluorescence detection and quantification during PCR amplification (real time) or at endpoint. Elimination of laborious post-PCR sample processing enables high-throughput analysis and greatly reduces the risk of carryover contamination. The Amplifluor primer system is a molecular switch for detecting DNA amplification by energy transfer between fluorophore and quencher (2)(3). The OFF-to-ON transition occurs when the conformation of the Amplifluor primer changes from “closed” intramolecular stem-loop structure to an “open” extended structure. This structural change is achieved when the Amplifluor primers …