Hybridization and Detection of Digoxigenin Probes on RNA Blots

Abstract

The probing of RNA gel blots (also called Northern blots) with labeled nucleic acids provides data on the relative levels of steady state gene expression, and on RNA processing. The principle of the technique was first described by Alwine et al. (1). The protocol described in this chapter demonstrates the use of nonradioactive digoxigenin-labeled probes (chapter 10 and chapter 11) on RNA blots (chapter 7 and chapter 8). The detection system employs an antidigoxigenin antibody/alkaline phosphatase conjugate, and the chemiluminescent substrate 3-(2′-spiroadamantane)-4-methoxy-4-(3″-phosphoryloxy)-phenyl-1,2-dioxetane (AMPPD) (2,3). The procedure used is similar to that described for probing Southern blots and detection by chemiluminescence in chapter 17. However, this procedure uses a different membrane for support of the immobilized nucleic acids. This, and the fact that RNA is being probed, require modifications to the hybridization buffer and washing procedure.