title: Semiquantitative Detection of Abnormal CD44 Transcripts in Colon Carcinomas by Reverse Transcription-Polymerase Chain Reaction Enzyme-linked Immunosorbant Assay (RT-PCR ELISA).
abstract: Background: Abnormal expression of CD44 variant RNA has been detected in a variety of human tumors and has been shown to be a potential diagnostic marker. To date, such analysis requires time-consuming gel electrophoresis, blotting, and autoradiographic procedures, and this approach may not be suitable for routine laboratory examinations. We have developed a rapid and semiquantitative reverse transcription-polymerase chain reaction enzyme-linked immunosorbent assay (RT-PCR ELISA) method and used it to analyze CD44 expression in colon carcinoma tissues and exfoliated cancer cells in colon luminal washings. Methods and Results: RNA was extracted from sample cells and tissues and converted to cDNA. PCR amplification products, labeled by incorporation of digoxigenin-11-dUTP, were hybridized with biotinylated probes complementary to CD44 exon 12 or to exons in the standard portion (CD44s) of the gene. Hybridized DNA complexes were immobilized on streptavidin-coated microtiter plates, and the bound PCR products were detected with a peroxidase-conjugated antibody to digoxigenin. CD44-derived PCR products were quantified by absorbance of a chromogenic reaction. Elevated expression of CD44 variant exon 12 was detected initially by Southern blot analysis in all of the 9 colon carcinoma tissues, while weak expression was observed in only 3 of 9 normal mucosas. This tumor-related differential expression was confirmed by the newly developed PCR-ELISA method. Elevated expression of CD44 exon 12 was also detected in exfoliated colonic epithelial cells from 10 of 13 carcinoma cases but not in exfoliated cells from 4 patients with inflammatory bowel disease. Conclusions: Raised expression of CD44 variant exon transcripts can be detected reliably in colonic tumor tissue and in exfoliated colonic cancer cells by a semiquantitative RT-PCR ELISA method. This was shown to be as sensitive as conventional RT-PCR using chemiluminescent detection. Therefore, CD44-based RT-PCR ELISA could facilitate detection of neoplasia in clinical specimens including colon washings and naturally micturated urine.
authors.affiliation: Nuffield Department of Pathology and Bacteriology, University of Oxford, Headington, Oxford, U.K.
authors.initials: K
authors.lastname: Yoshida
authors.initials: S
authors.lastname: Goodison
authors.initials: T
authors.lastname: Sugino
authors.initials: J
authors.lastname: Bolodeoku
authors.initials: M
authors.lastname: Churchman
authors.initials: BF
authors.lastname: Warren
authors.initials: D
authors.lastname: Tarin