Since the ligation and the purification steps for preparing cDNA are expected to play the central role in the origin of NSA, we surveyed the literature between 2017 -January 2021 for published methods. Among thirty-three reported bioanalytical RCA studies involving presynthesized cDNA, splint-padlock ligation was applied overwhelmingly (73%) followed by a distant blunt-ended ligation (15%, Figure ). For those involving splint-padlock ligation, the splint length varied between 20 to 40 nt with the majority of them in the 20 -25 nt region (63%, Figure ). The purification methods utilized either dual Exo I/Exo III digestions under five types of buffer conditions (48.5%), no digestion (or no information provided, 39.4%), PAGE purification (6.1%), or single enzyme (either Exo I or Exo III) digestion (6%) (Figure ). It was unclear (either qualitatively or quantitatively) which of these diverse ligations as well as purification steps could specifically help in having an NSA-free RCA assay. Besides, the rationale behind selecting the ligation or purification conditions remained ambiguous. Given its growing utilization as a bioanalytical assay, quantifying, understanding, and mitigating the NSA in RCA remains of utmost importance.