Our experiments quantitatively demonstrated that the non-specific amplification was generally the highest for cDNA prepared from splint-padlock mediated ligation (with 38 nt long splint), followed by cohesive end ligation, and usually the least for self-annealed ligation. In fact, throughout this study, self-annealing ligated cDNA displayed the generally minimal presence of NSA irrespective of digestion condition with its lowest NSA recorded for dual Exo I and III digestion in 1X Exo III buffer. The reproducible observation that splint-padlock ligation generates non-specific amplification even after exclusive Exo I or III digestion corroborates earlier findings by at least two independent groups . Its high degree of NSA for longer splint length was even immune to mitigation by application of a longer duration of snap-cooling. Rather, it demonstrated a splint-length dependent correlation with NSA, with decreasingly lower NSA observed with reducing splint lengths. Its least NSA was detected for 16 nt splint for dual exonuclease digestions with 3X Exo III buffer (having 33 mM Mg 2+ ) condition. Therefore, the high thermodynamic stability of the splint-cDNA association (for a longer splint) was identified as the possible cause behind the non-specific amplifications in it.