can any one provide tips or clues on RNA Ligase Mediated (RLM) 5' RACE? We have used dsRNA to cleave target mRNA transcript and are trying to use RLM 5' RACE to determine the cleavage sites of the mRNA.
First, we assume that dsRNA produces siRNA (18-25 nt, but maybe majority is 21-nt) which cleaves the target mRNA once or more times, then the cleaved mRNA goes through general RNA decay. Then I realize that this may not be the case because siRNA could cleave its target at multiple sites.
In either case, could it be feasible to use RLM 5 RACE to determine the cleavage sites? If there are multiple cleavage sites, we would expect multiple bands of cDNA on the gel. Or we may not detect bands due to the immediate degradation of cleaved mRNA or too many bands (like smear) on the gel.
So, which of the above would be the case(s)? We are quite new in using RLM 5 RACE to detect siRNA-mediated cleave.
I will greatly appreciate your help.