Rapid colorimetric detection of epidermal growth factor receptor mRNA by in situ hybridization

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Abstract
We describe a rapid, formamide‐free, random oligomer‐enhanced in situ hybridization method in formalin‐fixed, paraffin‐embedded tissue sections using a biotinylated oligonucleotide probe for colorimetric detection of the mRNA transcript of the epidermal growth factor receptor (EGFR) gene, a putative protooncogene. Transitional cell carcinomas (TCC) of the urinary bladder and oral squamous cell carcinomas (SCC) were examined. A431, a human squamous carcinoma cell line that overexpresses EGFR mRNA, and mature skeletal muscle, Known not to express EGFR, served as control tissues. A biotinylated poly‐T oligonucleotide probe was used to evaluate the preservation of mRNA in the formalin‐fixed, paraffin‐embedded tissues. The EGFR mRNA and poly‐T oligonucleotide probes were constructed with a target‐specific, 5′ region and a 3′, non‐hybridizing, biotinylated tail. Random sequence oligomers nine bases long added to the probe cocktail eliminated the need for formamide and sheared carrier DNA in the method. The assay produced strong, specific staining for EGFR mRNA in all cases of TCC and SCC, and in the A431 tumors, but not in skeletal muscle. The assay was completed in approximately 90 minutes. This method may have widespread utility for rapid and specific detection of other mRNA sequences.