NONRADIOACTIVE NORTHERN BLOTTING WITH BIOTINYLATED AND DIGOXIGENIN-LABELED RNA PROBES

The application of nonradioactive RNA probes for Northern blotting off ers the advantage of a rapid turn-around time for results without the loss of sensitivity for target mRNA detection. However, a problem that has impeded the widespread use of nonradioactive RNA probes for use i n Northern blotting is the difficulty in stripping these probes from n ylon membranes after hybridization. In this report we describe two pro tocols for stripping digoxigenin (Dig)labeled RNA probes from nylon me mbranes. One protocol utilizes a phosphate-buffered formamide strippin g solution to remove nonchemically modified (regular) RNA probes while the other method utilizes strippable probes that were produced with a chemically modified nucleotide (CTP) and removed by a specific stripp ing solution. This latter method was developed by Ambion Inc. and is c alled Strip-EZ(TM). We, also describe a protocol for the detection of two separate rat mRNAs using both biotin and digoxigenin-labeled RNA p robes that does not require stripping the membrane after hybridization . Finally, we describe the use of another new labeling technology, cal led Chem-Link(TM), that quickly and conveniently labels RNA for use in Northern blotting.