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

A simplified, nonradioactive procedure for the detection of specific m RNAs on Northern blots has been developed, utilizing digoxigenin-label led oligonucleotides and chemiluminescence. Antisense oligonucleotide (30-35 mer) probes were designed and synthesised based on published cD NA and gene sequences. These probes were end-labelled (5') with digoxi genin. Total RNA was fractionated by agarose gel electrophoresis and c apillary blotted onto positively charged nylon membranes. After hybrid ization, the mRNA/digoxigenin-labelled oligonucleotide complex was det ected by a chemiluminescence-based method using disodium 'chloro)-tric yclo[3.3.1.1(3,7)]decane]-4-yl)phenyl phosphate (CSPD) as substrate. T he advantages of this simplified technique for detecting mRNAs in phys iological and nutritional studies are described.