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Analysis of retinoic acid receptor beta expression in normal and malignant laryngeal mucosa by a sensitive and routine applicable reverse transcription-polymerase chain reaction enzyme-linked immunosorbent assay method.

L Castillo, G Milano, J Santini, F Demard and V Pierrefite
L Castillo
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G Milano
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J Santini
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F Demard
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V Pierrefite
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DOI:  Published November 1997
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Abstract

Retinoic acid receptor beta (RAR-beta) seems to be a useful intermediate marker in trials of retinoids, and the aim of this work was to describe a fast, sensitive, and routine applicable method to measure RAR-beta expression in tumor biopsies. We developed a new technique combining reverse transcription-PCR with a colorimetric ELISA detection of amplification products. The principle of this nonradioactive method is based on digoxigenin labeling of PCR products during amplification. Amplified DNA is hybridized with a biotinylated capture probe. The generated hybrid is immobilized on a streptavidin-coated microtiter plate, and detection is performed with the use of an antidigoxigenin peroxidase conjugate. We applied this method to quantify the expression of RAR-beta and an internal control (beta2 microglobulin) in laryngeal tumors. We found a detection threshold at 50 pg of PCR products, which represents a 100-fold improvement when compared to the detection limit of ethidium bromide detection. The method was reproducible (intra- and interassay reproducibilities at 7 and 5%, respectively). We used this technique for determining RAR-beta expression in 20 patients with laryngeal carcinoma and in 20 patients without cancer. The data show that the value of the RAR-beta:beta2 microglobulin ratio is decreased in tumoral versus nontumoral specimens (P = 0.0012), which is consistent with previously published results.

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November 1997
Volume 3, Issue 11
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Analysis of retinoic acid receptor beta expression in normal and malignant laryngeal mucosa by a sensitive and routine applicable reverse transcription-polymerase chain reaction enzyme-linked immunosorbent assay method.
L Castillo, G Milano, J Santini, F Demard and V Pierrefite
Clin Cancer Res November 1 1997 (3) (11) 2137-2142;

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Analysis of retinoic acid receptor beta expression in normal and malignant laryngeal mucosa by a sensitive and routine applicable reverse transcription-polymerase chain reaction enzyme-linked immunosorbent assay method.
L Castillo, G Milano, J Santini, F Demard and V Pierrefite
Clin Cancer Res November 1 1997 (3) (11) 2137-2142;
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Clinical Cancer Research
eISSN: 1557-3265
ISSN: 1078-0432

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