RAPID COLORIMETRIC DETECTION OF EPIDERMAL GROWTH-FACTOR RECEPTOR MESSENGER-RNA BY IN-SITU HYBRIDIZATION

We describe a rapid, formamide-free, random Oligomer-enhanced in situ hybridization method in formalin-fixed, paraffin-embedded tissue secti ons using a biotinylated oligonucleotide probe for colorimetric detect ion of the mRNA transcript of the epidermal growth factor receptor (EG FR) gene, a putative protooncogene. Transitional cell carcinomas (TCC) of the urinary bladder and oral squamous cell carcinomas (SCC) were e xamined. A431, a human squamous carcinoma cell line that overexpresses EGFR mRNA, and mature skeletal muscle, known not to express EGFR, ser ved as control tissues. A biotinylated poly-T oligonucleotide probe wa s used to evaluate the preservation of mRNA in the formalin-fixed, par affin-embedded tissues. The EGFR MRNA and poly-T oligonucleotide probe s were constructed with a target-specific, 5' region and a 3', non-hyb ridizing, biotinylated tail. Random sequence oligomers nine bases long added to the probe cocktail eliminated the need for formamide and she ared carrier DNA in the method. The assay produced strong, specific st aining for EGFR mRNA in all cases of TCC and SCC, and in the A431 tumo r, but not in skeletal muscle. The assay was completed in approximatel y 90 minutes. This method may have widespread utility for rapid and sp ecific detection of other mRNA sequences. (C) 1993 Wiley-Liss, Inc.