Markovic B, Kwan Y L, Nicholls E M, Walsh C, Crouch R L
Department of Haematology, Prince of Wales Hospital, Randwick, New South Wales, Australia.
J Pathol. 1992 Aug;167(4):369-73. doi: 10.1002/path.1711670404.
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.
我们开发了一种高灵敏度的非放射性原位杂交技术，该技术使我们能够研究组织中mRNA的产生。作为我们方法验证程序的一部分，我们检查了检测mRNA的聚腺苷酸（poly-A）RNA尾巴的各种方法。我们使用了三种与聚腺苷酸序列互补的生物素标记探针：一种25聚体的聚dT寡核苷酸、一种dT聚合物和一种dT:rA杂聚物。所有探针都具有相同的反应特异性，但通过使用碱性磷酸酶作为检测酶进行组织化学观察时，dT:rA杂聚物产生的信号最强。所有测试用于检测聚腺苷酸的探针均显示出反应性。聚dT寡核苷酸显示出与已发表结果相当的信号强度。生物素化的dT聚合物产生的信号比寡核苷酸更强，而杂聚物的信号最强。杂聚物探针产生的强信号是由于杂交过程中的探针复合，其中探针的正义链和反义链（即聚rA和聚dT）之间额外的结合增加了杂交位点处生物素分子的数量；我们已采用这种策略来可视化低拷贝数的特定mRNA。
