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Clinical Chemistry 47: 486-490, 2001;
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(Clinical Chemistry. 2001;47:486-490.)
© 2001 American Association for Clinical Chemistry, Inc.

Articles

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

Karl-Anton Kreuzer2,1, Alexander Bohn2,1, Joachim Lupberger1, Jerome Solassol1, Philipp le Coutre1 and Christian Andreas Schmidta,1

1 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.
a Author for correspondence. Fax 49-30-45053929; e-mail christian.schmidt{at}charite.de.

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 ß-actin molecules as control templates, 105 bcr/abl molecules were amplified, and 105 ß-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 10–105 copies/100 ng of cDNA. In the same PCR reactions, r = 0.99 for ß-actin (coamplified with M-bcr/abl or m-bcr/abl) for 103–107 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 ß-actin transcripts allows monitoring of bcr/abl+ leukemias.




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