Among the different digestion conditions explored, minimal non-specific amplification was observed only for dual Exo I and III digestion in presence of Exo III buffer (with or without Mg 2+ supplement). Interestingly, this has been only used in 3% of the surveyed papers. Another interesting finding was the almost complete absence of NSA during LRCA to a substantial increase during HRCA for cohesive end ligated cDNAs 11C -13C. A correlation with the length of cohesive end also seemed to indicate the NSA increased with successively lower cohesive end length. We could not compare or validate this observation with other published studies as the use of cohesive end ligation in bioanalytical RCA assays is sparse. A thrombin biosensing assay using a pre-synthesized cohesive end ligated cDNA utilized phenolchloroform purification (without any enzymatic digestion) but did not comment on the presence of NSA . A second report aiming to quantify NSA for cohesive end ligated cDNA utilized restriction enzyme generated 5-protruding sticky ends for the synthesis of dumbbell-shaped symmetric cDNA . Using the same cDNA without any further treatment, it then reported the absence of NSA for LRCA and circle-to-circle-RCA condition . While this observation seconded our finding of low NSA for LRCA condition, the quantification and then perplexing observation of high NSA under HRCA was unique to our study.