A NOVEL METHOD FOR REAL-TIME QUANTITATIVE RT PCR

A novel approach to quantitative reverse transcriptase polymerase chai n reaction (QC RT-PCR) using real time detection and the 5' nuclease a ssay has been developed. Cystic fibrosis transmembrane transductance r egulator (CFTR) target mRNA is reverse transcribed, amplified, detecte d, and quantitated in real time. A fluorogenic probe was designed to d etect the CFTR amplicon. Relative increase in 6-carboxy-fluorescein re porter fluorescent emission is monitored during PCR amplification usin g an analytical thermal cycler. An internal control template containin g the same primer sequences as the CFTR amplicon, but a different inte rnal sequence, has been designed as a control. An internal control pro be with a reporter fluorescent dye tetrachloro-6-carboxy-fluorescein w as designed to hybridize to the internal control amplicon. The interna l control template is placed in each reaction tube and is used for qua ntitative analysis of the CFTR mRNA. This method provides a convenient and high-throughput format for QC RT-PCR.