I've been doing miRNA northern bloting for over months. I have a question: Dose anybody know whether loading of total RNA with EB and run in denatured PAGE gel (15%, urea) cause shifting of the microRNA bands or not? I stained the gel post electrophoresis but the bands are poorly visualized; therefore I load total RNA with EB then run the gel, I found the signals of the rRNA and tRNA bands are very strong, but after hybridization of the transferred membrane, I found the band signals of the target miRNA as well as the artificial miRNA appeared in inappropriate position. They should be appeared in between the xylene cyanol and bromophenol blue bands. Could anybody tell me if I make it wrong? Why?
Ethtidium bromide makes double stranded DNA run about 10% slower than EB free dna so it is possible that the secondary structure of the RNA is intercalating EB which is slowing its migration down
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