Next, we investigated the NSA under hyperbranched RCA (HRCA) conditions for selfannealed and cohesive end ligation. During HRCA, a primer sharing the same sequence as part of the circular DNA hybridizes to LRCA amplicon at multiple sites and initiates strand displacement amplification (Scheme S1). Using this exponentially branching amplification, HRCA significantly improves the analytical sensitivity of otherwise linear RCA by several orders of magnitude, sometimes with a limit of detection as low as 100 -1000 copies . As a result, it is frequently employed in RCA assays involving both in situ ligation as well as pre-synthesized cDNA . Thanks to a highly sensitive quantitative fluorescence-based readout, the HRCA primer-assisted amplification of cDNA (but without an LRCA primer) would enhance an otherwise undetectable low quantity of NSA from LRCA condition. Given the frequent use of HRCA conditions in bioanalytical assays, it would therefore help probe the complete spectrum of the possibility of non-specific "false positive" RCA and in turn help in NSA quantification. For this investigation, HRCA primer concentration was chosen as 0.2 M in accordance with literature reported RCA assays involving in situ ligation and circular DNA . In these experiments, we had selected conditions C5 -C7 as other conditions (C1 -C4, C8, C9) already showed relatively higher end-RFU and NNSA for even self-annealing and cohesive end ligation (above). Similarly, HRCA primer assisted NSA in splint-padlock ligation was also not investigated at this stage as the NNSA in it remained high (at least 32%, in condition C7). A quantitative summary of these experiments has been provided in Table .