siRNA stability analysis by siRNA_profile and novel guidelines for highly efficient siRNA design. The average energy profiles of 2431 siRNA sequences  were calculated and analyzed by siRNA_profile program. The average energy profiles of siRNA sequences were divided into categories according to their efficiencies (GE categories: ≤ 20%, 20.1%–30%, 30.1%–40%, 40.1%–50%, 50.1%–70% and ≤70%). The siRNA efficiency showed siRNA instability dependency: highly functional siRNA molecules (GE ≤ 20%, group a) illustrated lower overall stability in addition to asymmetric average energy profile than moderately (GE 20.1 – 50%, group b) or non-functional (GE >50%, group c) siRNA molecules. Statistical analyses were performed with one-way ANOVA: (*) average internal stabilities of highly functional siRNAs (a) compared to moderately functional ones (b); Φ average internal stabilities of moderately functional siRNAs (b) compared to non-functional ones (c). ** p < 0.01; ***/ΦΦΦ p < 0.001.