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

J Pathol. 1989 Aug;158(4):279-86. doi: 10.1002/path.1711580403.

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 transferase 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 hybridization 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Biotin / analogs & derivatives
  • Colon / analysis
  • Deoxyuracil Nucleotides
  • Fixatives
  • Formaldehyde
  • Humans
  • Nucleic Acid Hybridization*
  • Oligonucleotides*
  • Palatine Tonsil / analysis
  • Paraffin
  • Poly A / analysis*
  • Poly T*
  • Polydeoxyribonucleotides*
  • RNA / analysis*
  • RNA Probes*
  • RNA, Messenger
  • Tissue Preservation

Substances

  • Deoxyuracil Nucleotides
  • Fixatives
  • Oligonucleotides
  • Polydeoxyribonucleotides
  • RNA Probes
  • RNA, Messenger
  • Formaldehyde
  • Poly A
  • Poly T
  • RNA
  • Biotin
  • Paraffin
  • biotin-11-dUTP