Bioanalytical RCA techniques involving pre-synthesized cDNA would not require in situ ligation. These assays therefore in principle are faster compared to those involving in situ ligation and have seen applications in the biosensing of viruses, microRNA detections, and imaging . A non-specific amplification or NSA is defined as the amplification in the absence of a target, and its origin in other isothermal amplifications such as LAMP and RPA has been traced to cross-primer annealing , pH , non-specific reporter dye binding , and nonspecific enzyme binding among other factors . Like other nucleic acid amplification methods, a bioanalytical RCA assay involving a pre-synthesized cDNA must also display minimal or none at all NSA. Two such possible sources of NSA could be inefficiencies at the ligation step preparing cDNA or its purification via denaturing gel purification or exonuclease digestion of precursor oligonucleotides. Even a trace amount of unligated precursor oligonucleotides from these two steps may hypothetically anneal to a pre-synthesized cDNA to cause NSA. Therefore, it is of paramount importance to ensure that there is no NSA associated with assays involving pre-synthesized cDNA. The NSA is difficult to distinguish from genuine, target-initiated RCA at a low target concentration range, may interfere with the limit of detection studies, and would risk generating false-positive readout. If not mitigated in RCA-based imaging experiments , such NSA could potentially contribute to background fluorescence. Although denaturing PAGE purification method should produce high purity cDNA free of NSA , it is low yield, laborious, and time-taking. In prior studies, the NSA has been detected by fluorescence measurement during real-time RCA or by Cas12a-mediated optomagnetic internal readout control . However, its removal or its mechanistic origin remained unprobed in both of these studies. In a study concerning cDNA preparation from restriction enzyme generated cohesive end ligation , exclusive Exonuclease I (Exo I) or Exonuclease III (Exo III) digestion alone failed to mitigate NSA from splint-padlock ligation. The same study reported NSA-free RCA on symmetric dumbbell-shaped cDNA prepared by restriction endonuclease generated cohesive end ligation. However, the quantitative contribution of exonuclease digestion in implementing non-specific amplification remained unaddressed in the study. Moreover, it is unknown (to the best of the authors' knowledge) whether the exonuclease digestion step (or the lack of it) has similar or different effects on the non-specific amplification in cDNA prepared from the self-annealed, cohesive end, and splint-padlock ligation.