Semiquantitative detection of abnormal CD44 transcripts in colon carcinomas by reverse transcription-polymerase chain reaction enzyme-linked immunosorbant assay (RT-PCR ELISA)

Kazuhiro Yoshida, Steven Goodison, Takashi Sugino, John Bolodeoku, Michael Churchman, Bryan F. Warren, David Tarin

Research output: Contribution to journalArticle

4 Citations (Scopus)

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-11dUTP, 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.

Original languageEnglish (US)
Pages (from-to)167-173
Number of pages7
JournalMolecular Diagnosis
Volume1
Issue number3
DOIs
StatePublished - Jan 1 1996
Externally publishedYes

Fingerprint

Reverse Transcription
Colon
Carcinoma
Polymerase Chain Reaction
Exons
Enzymes
Digoxigenin
Neoplasms
Enzyme-Linked Immunosorbent Assay
RNA
Streptavidin
Southern Blotting
Inflammatory Bowel Diseases
Colonic Neoplasms
Peroxidase
Electrophoresis
Mucous Membrane
Complementary DNA
Gels
Epithelial Cells

Keywords

  • CD44
  • Colon carcinoma
  • ELISA
  • Noninvasive detection of cancer
  • RT-PCR

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Semiquantitative detection of abnormal CD44 transcripts in colon carcinomas by reverse transcription-polymerase chain reaction enzyme-linked immunosorbant assay (RT-PCR ELISA). / Yoshida, Kazuhiro; Goodison, Steven; Sugino, Takashi; Bolodeoku, John; Churchman, Michael; Warren, Bryan F.; Tarin, David.

In: Molecular Diagnosis, Vol. 1, No. 3, 01.01.1996, p. 167-173.

Research output: Contribution to journalArticle

Yoshida, Kazuhiro ; Goodison, Steven ; Sugino, Takashi ; Bolodeoku, John ; Churchman, Michael ; Warren, Bryan F. ; Tarin, David. / Semiquantitative detection of abnormal CD44 transcripts in colon carcinomas by reverse transcription-polymerase chain reaction enzyme-linked immunosorbant assay (RT-PCR ELISA). In: Molecular Diagnosis. 1996 ; Vol. 1, No. 3. pp. 167-173.
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AU - Sugino, Takashi

AU - Bolodeoku, John

AU - Churchman, Michael

AU - Warren, Bryan F.

AU - Tarin, David

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AB - 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-11dUTP, 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.

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