CHEMILUMINESCENT DETECTION OF MESSENGER-RNAS ON NORTHERN BLOTS WITH DIGOXIGENIN END-LABELED OLIGONUCLEOTIDES

To establish a simplified, nonradioactive approach for identifying mRN As on Northern blots, antisense oligonucleotides have been used as pro bes in combination with chemiluminescence-based detection. Oligonucleo tides (similar to 32-mer) were end-labeled with digoxigenin (DIG) and used in conjunction with adamantyl 1,2-dioxetane aryl phosphate substr ates (Lumigen PPD and CSPD). Oligonucleotides were designed as probes for several mRNAs in tissues of rats and mice, including the mitochond rial uncoupling protein, lipoprotein lipase, GLUT1, GLUT4, and beta-ac tin. Uncoupling protein mRNA was detected in total RNA from brown adip ose tissue with a 32-mer DIG-labeled oligo-nucleotide, within 2 min of exposure to film. This mRNA could also be detected when as little as 250 ng of total RNA was applied to the gel, following 4 h exposure to film, and was present only in brown fat. The mRNA for lipoprotein lipa se was detectable with a 30-mer DIG-labeled oligonucleotide in 1 mu g of total RNA from mouse heart, within 2 h of exposure. The mRNA for th e GLUT1 glucose transporter was detected in total RNA from rat midbrai n using a 32-mer DIG-labeled oligonucleotide, while beta-actin mRNA wa s detected with a 30-mer oligonucleotide. The mRNA for the insulin-sen sitive glucose transporter GLUT4 was detected with a 32-mer DIG-labele d oligonucleotide and found only in those tissues in which glucose upt ake is stimulated by insulin. The speed of detection was greater with CSPD and was augmented by exposure of membranes to film at 37 degrees C. It is suggested that DIG-labeled oligonucleotides, in combination w ith chemiluminescence detection, can provide a rapid, sensitive, nonra dioactive procedure for probing mRNAs on Northern blots. (C) 1994 Acad emic Press, Inc.