In situ hybridization demonstration of poly-adenylated RNA sequences in formalin-fixed paraffin sections using a biotinylated oligonucleotide poly d(T) probe.
J Pathol
; 158(4): 279-86, 1989 Aug.
Article
en En
| MEDLINE
| ID: mdl-2475601
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.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Oligonucleótidos
/
Poli A
/
Poli T
/
Polidesoxirribonucleótidos
/
ARN
/
Sondas ARN
/
Hibridación de Ácido Nucleico
Límite:
Humans
Idioma:
En
Revista:
J Pathol
Año:
1989
Tipo del documento:
Article
Pais de publicación:
Reino Unido