Analysis of yeast trimethylguanosine-capped RNAs by midwestern blotting.
Immuno-detection by 'Midwestern' blotting provides a simple way to identify trimethylguanosine (TMG) capped RNAs. With this technique, over 20 bands are observed when total cellular RNA from Saccharomyces cerevisiae is transferred to a nylon membrane and probed with anti-TMG antibodies. Most, if not all, species known to contain a TMG cap are detected by this method. Only TMG-capped RNAs are detected on Midwestern blots unlike anti-TMG immunoprecipitates. Midwestern blotting is a useful alternative to immunoprecipitation and Northern analysis and may prove to be a better method for determining the relative abundance of capped RNAs. The blots can be reprobed multiple times with labeled antisense oligonucleotides to determine the identity of any TMG-capped species for which the primary sequence or a clone is available. This dual detection capability provides a powerful tool for the analysis of TMG-capped snRNAs and snoRNAs.
Rasmussen
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TP
Laboratories of Genetics and Molecular Biology, University of Wisconsin, Madison 53706, USA.
Culbertson
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MR
D000906:Antibodies / Q000276:immunology / Q000378:metabolism; D015152:Blotting, Northern; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics; D016373:DNA, Antisense / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006151:Guanosine / Q000031:analogs & derivatives*; D015151:Immunoblotting; D012315:RNA Caps / Q000032:analysis*; D012331:RNA, Fungal / Q000032:analysis; D012342:RNA, Small Nuclear / Q000032:analysis; D012441:Saccharomyces cerevisiae / Q000737:chemistry*
D000906:Antibodies; D015342:DNA Probes; D016373:DNA, Antisense; D012315:RNA Caps; D012331:RNA, Fungal; D012342:RNA, Small Nuclear; D006151:Guanosine