In situ hybridization demonstration of poly‐adenylated RNA sequences in formalin‐fixed paraffin sections using a biotinylated oligonucleotide poly d(T) probe

Abstract

An in situ hybridization technique has been developed for assessing poly(A)⁺ RNA preservation in routine pathology specimens. The method detects poly-adenylated RNA sequences in tissue sections using a biotinylated polydeoxythymidine poly d(T)) probe. The probe was prepared from single-stranded 25–30 base oligo d(T) and was biotinylated using the enzyme terminal deoxynucleotide (ransferase with biotin-11-dUTP and dTTP in the ratio 1:4. The hybridization protocol uses varying concentrations of proteinase K to unmask mRNA sequences and the biotin labelled hybrids are demonstrated after hybridization under standard conditions by the application of streptavidin and biotinylated alkaline phosphatase. Alkaline phosphatase was visualized using a Fast Red naphthol-capture method and the sections were counterstained with haematoxylin. The results have confirmed that the method is specific for poly(A)⁺ RNA and shows that poly(A)⁺ RNA can be demonstrated in routine formalin-fixed sections using non-radioactive techniques with retention of morphology. It also provides a means of optimizing the hybritdization conditions for specific mRNA probes and produces a staining pattern demonstrating the relative level of poly(A)⁺ RNA per cell which may reveal new information about cell activity and tissue function.