A novel method for real-time quantitative RT-PCR has been developed using a 5' nuclease assay and real-time detection. This method allows for the quantification of CFTR target mRNA in real time. A fluorogenic probe was designed to detect the CFTR amplicon, and an internal control template was used to ensure accurate quantitation. The method provides a convenient and high-throughput format for quantitative RT-PCR. The polymerase chain reaction (PCR) is a rapid and powerful technique for the in vitro amplification of DNA. Quantitative PCR (qPCR) has been used to quantitate small amounts of DNA, and quantitative RT-PCR has been used to measure mRNA. qPCR and quantitative RT-PCR methods use an internal control that is coamplified with the target sequence. The internal control can be designed in several ways, including scrambling of the internal sequence, mutation of the target amplicon, deletion or insertion of sequences into the target amplicon, or splicing of the target primer sequences onto a nonhomologous DNA sequence. The 5' nuclease assay for detecting PCR products uses a nonextendable oligonucleotide hybridization probe labeled with a reporter fluorescent dye at the 5' end and a quencher fluorescent dye at the 3' end. When the probe is intact, the reporter dye emission is quenched. During the extension phase of the PCR cycle, the nucleolytic activity of the DNA polymerase cleaves the hybridization probe and releases the reporter dye. The resulting relative increase in reporter fluorescent dye emission is monitored in real time during PCR amplification using a sequence detector. The use of a charge coupled device (CCD) camera permits the detection of a wide spectrum of emission wavelengths. By using target and control probes containing different reporter fluorescent dyes, it is possible to detect both target and control RNA in a single reaction tube. However, this approach is limited to concentrations of target and internal control RNA or DNA that are within 1000-fold of each other. The method described allows for the quantification of CFTR target mRNA in real time. The use of a known amount of internal control RNA ensures accurate quantitation. The method provides a dynamic range of over 10^6.5 target molecules and has excellent precision with intra-assay CVs of <2% and interassay CVs of <3%. The method is sensitive, accurate, and can be used to quantitate large numbers of samples in a relatively short time. The use of two different fluorescent hybridization probes and a known concentration of internal control RNA allows the initial mRNA copy number of an unknown target to be calculated.A novel method for real-time quantitative RT-PCR has been developed using a 5' nuclease assay and real-time detection. This method allows for the quantification of CFTR target mRNA in real time. A fluorogenic probe was designed to detect the CFTR amplicon, and an internal control template was used to ensure accurate quantitation. The method provides a convenient and high-throughput format for quantitative RT-PCR. The polymerase chain reaction (PCR) is a rapid and powerful technique for the in vitro amplification of DNA. Quantitative PCR (qPCR) has been used to quantitate small amounts of DNA, and quantitative RT-PCR has been used to measure mRNA. qPCR and quantitative RT-PCR methods use an internal control that is coamplified with the target sequence. The internal control can be designed in several ways, including scrambling of the internal sequence, mutation of the target amplicon, deletion or insertion of sequences into the target amplicon, or splicing of the target primer sequences onto a nonhomologous DNA sequence. The 5' nuclease assay for detecting PCR products uses a nonextendable oligonucleotide hybridization probe labeled with a reporter fluorescent dye at the 5' end and a quencher fluorescent dye at the 3' end. When the probe is intact, the reporter dye emission is quenched. During the extension phase of the PCR cycle, the nucleolytic activity of the DNA polymerase cleaves the hybridization probe and releases the reporter dye. The resulting relative increase in reporter fluorescent dye emission is monitored in real time during PCR amplification using a sequence detector. The use of a charge coupled device (CCD) camera permits the detection of a wide spectrum of emission wavelengths. By using target and control probes containing different reporter fluorescent dyes, it is possible to detect both target and control RNA in a single reaction tube. However, this approach is limited to concentrations of target and internal control RNA or DNA that are within 1000-fold of each other. The method described allows for the quantification of CFTR target mRNA in real time. The use of a known amount of internal control RNA ensures accurate quantitation. The method provides a dynamic range of over 10^6.5 target molecules and has excellent precision with intra-assay CVs of <2% and interassay CVs of <3%. The method is sensitive, accurate, and can be used to quantitate large numbers of samples in a relatively short time. The use of two different fluorescent hybridization probes and a known concentration of internal control RNA allows the initial mRNA copy number of an unknown target to be calculated.