Simultaneous absolute quantification of target and control templates by real-time fluorescence reverse transcription-PCR using 4-(4 '-dimethylaminophenylazo)benzoic acid as a dark quencher dye

Background: Despite the many advantages of real-time fluorescence reverse t ranscription-PCR (RT-PCR) as a quantitative analytical tool, simultaneous q uantification of target and reference templates within one reaction has not been reported. We developed such an assay with an internal reference templ ate. Methods: For quantification of target and reference sequences, we used two fluorescent probes in one reaction vessel on an ABI PRISM 7700 SDS instrume nt. Fluorescent probes were labeled with either 6-carboxy-fluorescein or he xachloro-6-carboxy-fluorescein as reporter dye and 4-(4'-dimethylaminopheny lazo)benzoic acid (DABCYL) as a dark quencher fluorophore. To test the sens itivity and specificity of this assay, serial dilutions of reference and ta rget templates were analyzed in one PCR reaction. In the presence of 10 bet a -actin molecules as control templates, 10(5) bcr/abl molecules were ampli fied, and 10(5) beta -actin molecules were amplified in the presence of 10 bcr/abl copies. We also performed single and duplex measurements on samples from five patients with documented Philadelphia chromosome-positive chroni c myelogenous leukemia disease courses (72 samples) and three with minor bc r/abl(+) acute myelogenous leukemias (26 samples). Results: For M-bcr/abl duplex RT-PCR, the correlation coefficient (r) for s tarting template amounts and threshold cycle values was 0.99; for m-bcr/abl , r = 0.96, indicating a precise log-linear relation for 10-10(5) copies/10 0 ng of cDNA. In the same PCR reactions, r = 0.99 for beta -actin (coamplif ied with M-bcr/abl or m-bcr/abl) for 10(3)-10(7) copies/100 ng cDNA. The li near correlation coefficient for single and duplex measurements was 0.98 fo r M- and m-bcr/abl in patient samples. Conclusions: DABCYL can be used as dark quencher fluorophore in real-time f luorescence PCR. The duplex fluorescence RT-PCR assay for bcr/abl and beta -actin transcripts allows monitoring of bcr/abl(+) leukemias. (C) 2001 Amer ican Association for Clinical Chemistry.