We compared our observation with prior literature reports of RCA bioanalytical studies involving pre-synthesized cDNA during the period January 2017 -2020 (Figure ). Our studies revealed that cDNA preparation using self-annealing ligation generally showed the least non-specific amplification (NSA) among the three types of stated ligation techniques. Surprisingly, it is one of the least explored methods for RCA bioanalytical assays using presynthesized cDNA in literature (Figure ). Secondly, it was clear from our studies that splintpadlock ligation has been inherently having the highest NSA for 24 nt or longer splint lengths. Similar to our finding, NSA persisted even after single exonuclease digestion by either Exo I or III for a cDNA generated from splint (20 nt)-padlock ligation but was removed after gel purification of cDNA . Yet it remains the most used method in preparing cDNA (Figure ).