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

Clin Cancer Res. 1997 Nov;3(11):2137-42.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Aged
  • Base Sequence
  • Enzyme-Linked Immunosorbent Assay
  • Humans
  • Laryngeal Neoplasms / genetics
  • Laryngeal Neoplasms / metabolism
  • Laryngeal Neoplasms / pathology*
  • Laryngeal Neoplasms / surgery
  • Larynx / metabolism
  • Larynx / pathology*
  • Middle Aged
  • Molecular Sequence Data
  • Mucous Membrane / metabolism
  • Mucous Membrane / pathology
  • Neoplasm Staging
  • Receptors, Retinoic Acid / analysis*
  • Receptors, Retinoic Acid / genetics*
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sequence Alignment
  • beta 2-Microglobulin / genetics

Substances

  • Receptors, Retinoic Acid
  • beta 2-Microglobulin
  • retinoic acid receptor beta