Nonradioactive Northern blot analysis of plant RNA and the application of different haptens for reprobing.

Northern blot analysis using radioactive probes is still the most common technique to determine the accumulation of transcripts in cells and tissues. The main disadvantages of this technique are the possible health hazard, inconvenience during handling, the high amount of RNA target necessary for detection, and difficulties with stripping and reprobing. In this paper, we propose an easily applicable protocol for Northern blot analysis of plant RNA with enhanced sensitivity using digoxigenin-, fluorescein-, or biotin-labeled in vitro transcripts derived from PCR products.

Experimental procedure for the detection of a rare human mRNA with the DIG System.

Newcomers to the DIG System often inquire about the possibility of performing Northern blot hybridizations with nonradioactive techniques. With the following examples, we would like to share our protocol for performing highly sensitive Northern blots. This procedure strictly adheres to the standard procedures detailed in our manuals and pack inserts, and there are no special "tricks" required.