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 transcription-PCR (RT-PCR) as a quantitative analytical tool, simultaneous quantification of target and reference templates within one reaction has not been reported. We developed such an assay with an internal reference template.


METHODS
For quantification of target and reference sequences, we used two fluorescent probes in one reaction vessel on an ABI PRISM 7700 SDS instrument. Fluorescent probes were labeled with either 6-carboxy-fluorescein or hexachloro-6-carboxy-fluorescein as reporter dye and 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL) as a dark quencher fluorophore. To test the sensitivity and specificity of this assay, serial dilutions of reference and target templates were analyzed in one PCR reaction. In the presence of 10 beta-actin molecules as control templates, 10(5) bcr/abl molecules were amplified, 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 chronic myelogenous leukemia disease courses (72 samples) and three with minor bcr/abl+ acute myelogenous leukemias (26 samples).


RESULTS
For M-bcr/abl duplex RT-PCR, the correlation coefficient (r) for starting 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/100 ng of cDNA. In the same PCR reactions, r = 0.99 for beta-actin (coamplified with M-bcr/abl or m-bcr/abl) for 10(3)-10(7) copies/100 ng cDNA. The linear correlation coefficient for single and duplex measurements was 0.98 for M- and m-bcr/abl in patient samples.


CONCLUSIONS
DABCYL can be used as dark quencher fluorophore in real-time fluorescence PCR. The duplex fluorescence RT-PCR assay for bcr/abl and beta-actin transcripts allows monitoring of bcr/abl+ leukemias.
Abteilung für Innere Medizin und Poliklinik m.S. Hämatologie und Onkologie, Campus Virchow-Klinikum, Medizinische Fakultät Charité der Humboldt-Universität zu Berlin, Augustenburger Platz 1, 13353 Berlin, Germany.
K A
KA
Kreuzer
A
A
Bohn
J
J
Lupberger
J
J
Solassol
P
P
le Coutre
C A
CA
Schmidt
D005456:Fluorescent Dyes; D016044:Fusion Proteins, bcr-abl / Q000032:analysis; D006801:Humans; D007938:Leukemia / Q000097:blood / Q000235:genetics; D010677:Philadelphia Chromosome; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013050:Spectrometry, Fluorescence; D014407:Tumor Cells, Cultured; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives*
D005456:Fluorescent Dyes; C063127:4-(4-dimethylaminophenylazo)benzoic acid; D004124:p-Dimethylaminoazobenzene; D016044:Fusion Proteins, bcr-abl