RNAse Protections

[Tim Angelotti]

I have a few practical questions about RNAse protection assays, for the 
experts.  I am trying to see if a deletion mutant that I cloned by PCR is 
real using a probe that spans the deletion.  I have found both a full 
length band and a band corresponding to a cut at the site of the 
deletion.  My question is how can I be sure that the cut at the deletion 
site is due to the presence of an alternative transcript and not due to 
secondary structure?  I am using Ambion's RPA II kit, hybridizing at 45 C 
overnight.  The band is of the right size, but I am concerned that it may 
be artifact.  Is this common, and would a higher temperature sort it out?