*2.2.1. Presence of Secondary structure*

It has been suggested that presence of local secondary structures (stem loops) in the target site restricts its accessibility to RISC and hence reduces the efficiency of the siRNA. So it is necessary to filter out those potential inaccessible target sites with strong secondary structures. The prediction of local secondary structure can be made by numerous RNA secondary structure prediction tools or packages like Mfold [7] or Vienna RNA package [8] that mainly predict minimum free energy secondary structure of a RNA sequence.
