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Rapid chemiluminescent detection of mRNAs on northern blots with digoxigenin end-labelled oligonucleotides
P Trayhurn1, J S Duncan, A Nestor
1Division of Biochemical Sciences, Rowett Research Institute, Bucksburn, Aberdeen, UK.
Abstract:
A simplified, nonradioactive procedure for the detection of specific mRNAs on Northern blots has been developed, utilizing digoxigenin-labelled oligonucleotides and chemiluminescence. Antisense oligonucleotide (30-35 mer) probes were designed and synthesised based on published cDNA and gene sequences. These probes were end-labelled (5') with digoxigenin. Total RNA was fractionated by agarose gel electrophoresis and capillary blotted onto positively charged nylon membranes. After hybridization, the mRNA/digoxigenin-labelled oligonucleotide complex was detected by a chemiluminescence-based method using disodium 3-(4-methoxyspiro-[1,2-dioxetane-3-2'(5'chloro)- tricyclo[3.3.1.13.7]decane]-4-yl)phenyl phosphate (CSPD) as substrate. The advantages of this simplified technique for detecting mRNAs in physiological and nutritional studies are described.