In situ hybridization demonstration of poly-adenylated RNA sequences in formalin-fixed paraffin sections using a biotinylated oligonucleotide poly d(T) probe.
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.
Pringle
J H
JH
Department of Pathology, Leicester Royal Infirmary, U.K.
Primrose
L
L
Kind
C N
CN
Talbot
I C
IC
Lauder
I
I
D001483:Base Sequence; D001710:Biotin / Q000031:analogs & derivatives; D003106:Colon / Q000032:analysis; D003856:Deoxyuracil Nucleotides; D005404:Fixatives; D005557:Formaldehyde; D006801:Humans; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides; D014066:Palatine Tonsil / Q000032:analysis; D010232:Paraffin; D011061:Poly A / Q000032:analysis*; D011071:Poly T; D011089:Polydeoxyribonucleotides; D012313:RNA / Q000032:analysis*; D015347:RNA Probes; D012333:RNA, Messenger; D014021:Tissue Preservation
D003856:Deoxyuracil Nucleotides; D005404:Fixatives; D009841:Oligonucleotides; D011089:Polydeoxyribonucleotides; D015347:RNA Probes; D012333:RNA, Messenger; D005557:Formaldehyde; D011061:Poly A; D011071:Poly T; D012313:RNA; D001710:Biotin; D010232:Paraffin; C045931:biotin-11-dUTP