A rapid colorimetric in situ messenger RNA hybridization technique for analysis of epidermal growth factor receptor in paraffin-embedded surgical specimens of human colon carcinomas.
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 tissue, it allows for retrospective analyses of human tumor specimens using archival material.
Radinsky
R
R
Department of Cell Biology, University of Texas M.D. Anderson Cancer Center, Houston 77030.
Bucana
C D
CD
Ellis
L M
LM
Sanchez
R
R
Cleary
K R
KR
Brigati
D J
DJ
Fidler
I J
IJ
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002277:Carcinoma / Q000737:chemistry*; D002294:Carcinoma, Squamous Cell / Q000737:chemistry; D003110:Colonic Neoplasms / Q000737:chemistry*; D003124:Colorimetry; D066246:ErbB Receptors / Q000032:analysis / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization / Q000379:methods*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D066246:ErbB Receptors