Using a cationic carbocyanine dye to assess RNA loading in Northern gel analysis.
In Northern blotting, one must have a means of assessing the uniformity of RNA loaded into each lane of a gel. As an alternative to "common gene" controls and previously published nucleic acid dyes (ethidium bromide, acridine orange, methylene blue), we have utilized a cationic carbocyanine dye (Stains All) for the assessment of RNA gel loading uniformity over the range of 5-25 micrograms RNA/lane. The following protocol is suitable for messages of well-characterized mobility and utilizes xylene cyanol as a 4-kb marker; as such, it will migrate between 28S and 18S rRNA over a wide range of agarose concentrations. Optimally, it is best that the message(s) of interest should migrate either as a smaller species than 18S or as a larger species than 28S; this allows either the 28S or 18S ribosomal band to be separated from the message(s) of interest by severing the gel transversely at the xylene cyanol front. Severing the gel in such a manner makes it possible to simultaneously submit that portion of the gel containing either the 28S or 18S rRNA band to Stains All staining while immediately continuing with the transfer of that portion of the gel containing the mRNA of interest. We have found the dye to interact linearly with rRNA whether data were gathered by densitometrically scanning the gels themselves or photographs of the gels.
Wade
M F
MF
Department of Physiology and Endocrinology Medical College of Georgia, Augusta 30912.
O'Conner
J L
JL
D000818:Animals; D015152:Blotting, Northern / Q000379:methods*; D002232:Carbocyanines; D003720:Densitometry; D005069:Evaluation Studies as Topic; D005260:Female; D012313:RNA / Q000032:analysis*; D051381:Rats; D013194:Staining and Labeling
D002232:Carbocyanines; D012313:RNA; C012040:stains-all