Short technical reports. Quantification of ribozyme-mediated RNA cleavage using silver-stained polyacrylamide gels.
A very sensitive, nonradioactive method for the detection of ribozymes, targets for ribozyme cleavage and cleavage products with subsequent densitometric quantification of RNA, has been developed. Amounts as low as 15 ng--corresponding to 0.16 pmol of a 300-nucleotide RNA molecule--can be visualized in denaturating polyacrylamide (PA) gels. This sensitive method allows analysis and quantification of ribozyme-mediated cleavage of in vitro-transcribed short stretches of RNA. The substitution of N'N'-methylene bisacrylamide by piperazine diacrylamide in the PA gels to improve signal-to-noise ratio could not further optimize the contrast. Sensitivity was dependent on the presence of urea in the gel and could be increased 4-fold in a gel without urea.
Palfner
K
K
Universitätsklinikum Göttingen, Germany.
Kneba
M
M
Hiddemann
W
W
Bertram
J
J
D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D012313:RNA / Q000032:analysis*; D016337:RNA, Catalytic / Q000378:metabolism*; D016622:Silver Staining
D016337:RNA, Catalytic; D012313:RNA