Hi
As can be seen on the attached gel image what or why might be occurring in well 2 (naked siRNA) that I am seeing 2 clearly distinct bands for what should be a one band migration of my 21bp pure siRNA. The other wells to the right are peptide/siRNA complexes in increasingly higher molar ratio.
Any ideas or explanations of what might be happening in the naked siRNA migration in well 2 to cause 2 bands rather than 1. I am ruling out RNase activity as there is no smearing of the RNA.
Gel is 3% agarose in 0.5 TBE. 70V for 60min. GELRED staining
Taking into account non-denaturing conditions of TBE phoresis, I guess that this can be an artifact of secondary structure or annealing.
In theory, some kinds of RNA are also very susceptible to spontaneous hydrolysis - typically one can't see 28S band from dipterans RNA samples since these large RNA's are cleaved to pieces merging with 18S RNA band. Unfortunately, I can't say what are the typical sequence or structure features of such self-cleaving RNA's. I'm not sure that they can be so short.
Trevor,
You may not see the smear because you loaded a pure sirna sample, however it appears to be partially degraded probably due to either some residual RNase activity left on the plastic or increasing the buffer/gel temperature during the electrophoresis. Also, if you preheated the samples before loading, it might've caused some degradation of the pure RNA samples..
You might want to check the sequence of the siRNA for several base-long stretches that may form a secondary structure (reverse complementarity) or that may lead to annealing of two molecules due to reverse complementarity . Actually, this is what I would guess given the nice clear bands: that you have a dimer formation.
Sven Buelow Irina Donahue Alexander Nesmelov . Thankyou Sven, Irina and Alexander. Your comments have been very helpful and have given me some ideas of what to think about. I have been in contact with the GELRED supplier and they have recommended that I use post gel staining as the pre gel staining method I have used might be causing some artefact of siRNA and GELRED and cause the additional band. I will this week try post gel staining and post the results back on here to give you some feedback. Thanks for all your help. Trevor
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