Next, we investigated strategies for splint-cDNA complex destabilization by shortening the splint length. The splint-padlock ligations in this work have so far involved 38 nt splint length, postulating that a longer splint would help efficient splint-padlock complex formation (through thermodynamic stability), thus increasing ligation yield. However, as evident so far, the long splint length was probably also contributory to high NSA in splint-padlock ligation. To quantify the effect of the splint length, we, therefore, utilized 30, 24, and 16 nt long splints (oligonucleotides 6 -8, respectively) for ligation using the same padlock sequence 4 as earlier.