Sensitive nonradioactive dot blot/ribonuclease protection assay for quantitative determination of mRNA.
Sensitive nonradioactive dot blot/ribonuclease protection assay for quantitative determination of mRNA.
复制标题
用于定量测定 mRNA 的灵敏非放射性斑点印迹/核糖核酸酶保护测定。
作者：
J. Zhan;H. Fahimi;A. Vöelkl
我们已经开发了一种简单而灵敏的方法，用于快速定量的mRNA从细胞培养物和小组织样品。该方法结合了核糖核酸酶保护试验的高灵敏度和特异性以及斑点印迹的简单操作和快速执行。地高辛标记的cRNA探针的使用消除了与核糖核酸酶保护测定中常用的放射性同位素相关的所有问题。RNA制备物直接点在尼龙膜上，杂交后用核糖核酸酶A处理滤膜，去除未杂交的单链RNA。然后使用标记的抗地高辛抗体通过化学发光技术使mRNA-杂交体可视化，并定量信号强度。与北方印迹核糖核酸酶保护测定法的比较显示，该斑点印迹技术的灵敏度几乎高出10倍，并且其信号在宽范围的RNA浓度（0.01-10微克/微升/斑点）内呈线性。这种方法似乎特别有价值的同时处理大量的样品含有广泛的RNA浓度。
We have developed a simple and sensitive method for the rapid quantitation of mRNA from cell cultures and small tissue samples. The method combines the high sensitivity and specificity of the ribonuclease protection assay with simple handling and rapid execution of dot blotting. The use of digoxygenin-labeled cRNA probes eliminates all problems associated with radioisotopes commonly used in the ribonuclease protection assay. The RNA preparation is dotted directly onto nylon membranes, and after hybridization the filters are treated with ribonuclease A, which removes the nonhybridized single-stranded RNA. The mRNA-hybrid is then visualized by the chemiluminescence technique using labeled anti-digoxigenin antibody, and the signal intensity is quantitated. Comparison with the Northern blotting ribonuclease protection assay revealed that this dot blot technique is almost ten times more sensitive and that its signals are linear over a wide range of RNA concentrations (0.01-10 micrograms/microL/dot). This method seems particularly valuable for simultaneous processing of large numbers of samples containing a wide range of RNA concentrations.