Nilsen Timothy W
Cold Spring Harb Protoc. 2015 Mar 2;2015(3):314-8. doi: 10.1101/pdb.prot081026.
Different laboratories use different methods for the prehybridization, hybridization, and washing steps of the northern blotting procedure. In this protocol, a northern blot is pretreated with Church and Gilbert hybridization buffer to block nonspecific probe-binding sites. The immobilized RNA is then hybridized to a DNA probe specific for the RNA of interest. Finally, the membrane is washed and subjected to autoradiography or phosphorimaging. The solutions and conditions described here may be ideal for those who prefer to use fewer ingredients in their solutions. This protocol is designed to achieve the same goals as other northern blotting approaches. It minimizes background (nonspecific adherence of probe to membrane and nonspecific hybridization) and maximizes specific hybridization to RNAs immobilized on a membrane.
不同实验室在Northern印迹法的预杂交、杂交和洗涤步骤中使用不同的方法。在本实验方案中，Northern印迹先用Church和Gilbert杂交缓冲液进行预处理，以封闭非特异性探针结合位点。然后将固定化的RNA与针对目标RNA的DNA探针杂交。最后，对膜进行洗涤并进行放射自显影或荧光成像。这里描述的溶液和条件对于那些希望在溶液中使用较少成分的人来说可能是理想的。本实验方案旨在实现与其他Northern印迹方法相同的目标。它将背景（探针与膜的非特异性结合以及非特异性杂交）降至最低，并使与固定在膜上的RNA的特异性杂交最大化。
