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Rapid chemiluminescent detection of mRNAs on Northern blots with digoxigenin end‐labelled oligonucleotides
Author(s) -
Trayhurn Paul,
Duncan Jacqueline S.,
Nestor Antoinette,
Thomas Moira E. A.,
Eastmond Nigel C.,
Rayner D. Ver
Publication year - 1995
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150160157
Subject(s) - digoxigenin , oligonucleotide , chemiluminescence , microbiology and biotechnology , complementary dna , blot , chemistry , biology , northern blot , agarose , rna , chromatography , messenger rna , biochemistry , dna , gene , in situ hybridization
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[3.3.1.1 3.7 ]decane]‐4‐yl)phenyl phosphate (CSPD) as substrate. The advantages of this simplified technique for detecting mRNAs in physiological and nutritional studies are described.