Luehrsen K R, Davidson S, Lee Y J, Rouhani R, Soleimani A, Raich T, Cain C A, Collarini E J, Yamanishi D T, Pearson J, Magee K, Madlansacay M R, Bodepudi V, Davoudzadeh D, Schueler P A, Mahoney W
Roche Diagnostics, Chief Technology Office, Berkeley, California 94710-2728, USA.
J Histochem Cytochem. 2000 Jan;48(1):133-45. doi: 10.1177/002215540004800114.
Oligonucleotides that carry a detectable label can be used to probe for mRNA targets in in situ hybridization experiments. Oligonucleotide probes (OPs) have several advantages over cDNA probes and riboprobes. These include the easy synthesis of large quantities of probe, superior penetration of probe into cells and tissues, and the ability to design gene- or allele-specific probes. One significant disadvantage of OPs is poor sensitivity, in part due to the constraints of adding and subsequently detecting multiple labels per oligonucleotide. In this study, we compared OPs labeled with multiple detectable haptens (such as biotin, digoxigenin, or fluorescein) to those directly conjugated with horseradish peroxidase (HRP). We used branching phosphoramidites to add from two to 64 haptens per OP and show that in cells, 16-32 haptens per OP give the best detection sensitivity for mRNA targets. OPs were also made by directly conjugating the same oligonucleotide sequences to HRP. In general, the HRP-conjugated OPs were more sensitive than the multihapten versions of the same sequence. Both probe designs work well both on cells and on formaldehyde-fixed, paraffin-embedded tissues. We also show that a cocktail of OPs further increases sensitivity and that OPs can be designed to detect specific members of a gene family. This work demonstrates that multihapten-labeled and HRP-conjugated OPs are sensitive and specific and can make superior in situ hybridization probes for both research and diagnostic applications.
携带可检测标记的寡核苷酸可用于原位杂交实验中探测mRNA靶标。与cDNA探针和核糖核酸探针相比，寡核苷酸探针（OP）具有多个优势。这些优势包括易于大量合成探针、探针能更好地穿透细胞和组织，以及能够设计基因或等位基因特异性探针。OP的一个显著缺点是灵敏度较差，部分原因是每个寡核苷酸添加并随后检测多个标记存在限制。在本研究中，我们将用多种可检测半抗原（如生物素、地高辛或荧光素）标记的OP与直接与辣根过氧化物酶（HRP）偶联的OP进行了比较。我们使用分支亚磷酰胺为每个OP添加2至64个半抗原，并表明在细胞中，每个OP 16至32个半抗原对mRNA靶标的检测灵敏度最佳。OP也通过将相同的寡核苷酸序列直接与HRP偶联来制备。一般来说，HRP偶联的OP比相同序列的多半抗原版本更灵敏。两种探针设计在细胞以及甲醛固定、石蜡包埋的组织上都能很好地发挥作用。我们还表明，OP混合物可进一步提高灵敏度，并且可以设计OP来检测基因家族的特定成员。这项工作表明，多半抗原标记的和HRP偶联的OP灵敏且特异，可为研究和诊断应用制备出更优质的原位杂交探针。
