Next, we explored possible measures to mitigate non-specific amplification (NSA) in splint-padlock ligated cDNA 4C which has consistently shown the highest NSA in C1 -C9 digestion conditions among the three substrates. We argued that this was due to strong splintpadlock complex formation preventing denaturation during snap-cooling as well as exonuclease digestion. As discussed earlier, early evidence of this was visible in denaturing PAGE where the post-ligation circular DNA (cDNA) 4C had slower mobility than the cDNA after exonuclease digestion (Figure ). It indicated that even denaturing gel conditions could only partially destabilize the splint-cDNA complex, reaffirming its high thermodynamic stability. Although exonuclease digestion increased the cDNA mobility, it was likely that the splint, probably in a partially digested form continued to remain attached to the cDNA. This was indirectly evident from the persistent large degree of NSA in all experiments involving splintpadlock ligation. We explored two strategies to destabilize the splint-cDNA association. In the first, aiming for a temperature-induced denaturation, we subjected the post-ligation solution to longer durations of snap cooling. To study the effect of temperature, snap cooling was carried out at 5, 10, and 15 min followed by dual digestion under C5 condition. However, pilot experiments involving LRCA conditions demonstrated that even longer snap-cooling (up to 15 min) did not help in mitigating NSA and continued to show 50% non-specific amplification (Figure ). Indicating that such temperature-induced destabilization has minimal effect on mitigating NSA, this was not replicated further for NNSA quantification LRCA or HRCA conditions.