Among C1 -C4, condition C3 (Exo I/III dual digestion with ligase buffer) usually displayed the least RFU and NNSA for all three substrates (Figure ). Among the three substrates, self-annealing ligated cDNA 1C has consistently demonstrated low end-RFU and NNSA compared to splint-padlock and cohesive end ligation for C1 -C4 conditions (Figure , Figure -D, Table ). In contrast, unusually high end-RFU in non-specific amplification and NNSA (1218.1 ± 163.5 end-RFU and 44.7 ± 13% NNSA for C3, the best condition among C1 -C4) was associated with splint-padlock ligated cDNA 4C for all conditions (Figure , Figure , Table ). The end-RFU and NNSA of cohesive end ligated cDNA 11C have remained intermediate between self-annealing ligation and splint-padlock ligation (Figure , Figure -L, Table ). In fact, conditions C1, C2, and C4 displayed relatively higher (compared to C3) high NNSA and high end-RFU for self-annealing as well as cohesive end ligation (Figure and). For splint padlock ligation, the NNSA showed higher than 100% RFU for conditions C1 and C4, indicating the non-specific amplification was even greater than primer assisted amplification (Figure , Table ). The relatively lower end-RFU and NNSA for C3 (compared to C1, C2, and C4) implied that the combined Exo I and III digestion would likely be more effective to minimize the NSA in all three types of ligations.