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Departments of Cell Biology [R. R., C. D. B., R. S., I. J. F.], Surgery [L. M. E.], and Pathology [K. R. C.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, and Department of Pathology, The University of Oklahoma Health Science Center, Oklahoma City, Oklahoma 73190 [D. J. B.]
We have developed a rapid colorimetric in situ mRNA hybridization procedure to analyze epidermal growth factor receptor (EGF-R) transcripts in paraffin-embedded surgical specimens of human colon carcinomas. This technique is based on the use of 24-base oligonucleotide probes labeled with 6 biotin molecules at the 3' end. mRNA integrity was verified using a hyperbiotinylated 30-residue-long deoxythymidylate oligonucleotide probe, and the specificity of the reaction was confirmed by using labeled EGF-R-specific sense and antisense probes. Avidin alkaline phosphatase detection and the capillary technology used in the Microprobe System allowed for completion of the procedure in under 5 h. The human A431 epidermoid carcinoma cells growing in culture and fixed with formalin as well as paraffin-embedded sections of this tumor growing s.c. in nude mice served as positive controls. In situ hybridization with antisense EGF-R oligonucleotide probes directly correlated with EGF-R mRNA and protein levels observed by Northern blot and immunohistochemistry, respectively. In situ hybridization of paraffin-embedded sections of primary human colon carcinoma and metastases from liver and lymph node revealed cell-specific staining with EGF-R antisense oligonucleotide probes that correlated directly with Northern blot and immunohistochemistry analyses. Since this rapid and sensitive in situ mRNA hybridization technique can be used in properly preserved paraffin-embedded tissues, it allows for retrospective analyses of human tumor specimens using archival material.
1 Supported in part by Cancer Center Support Core Grant CA 16672; Grant T32 CA 09599 and Grant R35 CA-42107 from the National Cancer Institute, NIH (I. J. F.); and Postdoctoral Fellowship Grant PF-3446 from The American Cancer Society (R. R.).
2 To whom requests for reprints should be addressed, at M. D. Anderson Cancer Center, Department of Cell Biology, HMB 173, 1515 Holcombe Boulevard, Houston, TX 77030.
3 Present address: UCLA Medical Center, Division of Surgical Oncology, 10833 Le Conte Avenue, Los Angeles, CA 90024.
Received 10/11/92. Accepted 1/19/93.
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