A sensitive method for the detection of poly-A tails of mRNA using a biotin-labelled heteropolymer of dT:rA.
We have developed a highly sensitive non-radioactive in situ hybridization technique that enables us to study the production of mRNAs in tissues. As part of the validation procedure of our methods, we examined various methods of detecting poly-A RNA tails of mRNA. We have used three types of biotin-labelled probes complementary to poly-A sequences: a 25-mer poly-dT oligonucleotide, a polymer of dT, and a heteropolymer of dT:rA. All the probes had the same specificity of reactivity but the heteropolymer of dT:rA gave the strongest signals as visualized histochemically by the use of alkaline phosphatase as the detection enzyme. All the probes tested for poly-A detection showed reactivity. The poly-dT oligonucleotide showed a strength of signal comparable to published results. The biotinylated polymer of dT gave a stronger signal than that of the oligonucleotide, and the heteropolymer was the strongest of all. The strong signal seen with the heteropolymer probe is due to probe complexing during hybridization, in which additional binding between sense and antisense strands of the probe (i.e. poly-rA and poly-dT) amplifies the number of biotin molecules at the hybridization site; this strategy has been exploited by us as a means of visualizing low copy numbers of specific mRNAs.
Department of Haematology, Prince of Wales Hospital, Randwick, New South Wales, Australia.
B
B
Markovic
Y L
YL
Kwan
E M
EM
Nicholls
C
C
Walsh
R L
RL
Crouch
D001853:Bone Marrow / Q000737:chemistry; D003106:Colon / Q000737:chemistry; D006801:Humans; D008198:Lymph Nodes / Q000737:chemistry; D009693:Nucleic Acid Hybridization; D011061:Poly A / Q000032:analysis*; D011071:Poly T; D015347:RNA Probes; D012333:RNA, Messenger / Q000032:analysis*
D015347:RNA Probes; D012333:RNA, Messenger; D011061:Poly A; D011071:Poly T