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Nonradioactive Northern blotting with biotinylated and digoxigenin‐labeled RNA probes
Author(s) -
Meltzer Jon C.,
Sanders Veronica,
Grimm Paul C.,
Chiasson Nathalie,
Hoeltke HansJoachim,
Garrett Karryn L.,
Greenberg Arnold H.,
Nance Dwight M.
Publication year - 1998
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.1150190825
Subject(s) - digoxigenin , rna , northern blot , blot , biotinylation , microbiology and biotechnology , membrane , chemistry , stripping (fiber) , molecular probe , nucleotide , messenger rna , biology , biochemistry , in situ hybridization , gene , materials science , composite material
The application of nonradioactive RNA probes for Northern blotting offers 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 in Northern blotting is the difficulty in stripping these probes from nylon membranes after hybridization. In this report we describe two protocols for stripping digoxigenin (Dig)‐labeled RNA probes from nylon membranes. One protocol utilizes a phosphate‐buffered formamide stripping 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 stripping solution. This latter method was developed by Ambion Inc. and is called Strip‐EZ™. We also describe a protocol for the detection of two separate rat mRNAs using both biotin and digoxigenin‐labeled RNA probes that does not require stripping the membrane after hybridization. Finally, we describe the use of another new labeling technology, called ChemLink™, that quickly and conveniently labels RNA for use in Northern blotting.