We argued that the quantification of the non-specific amplification (NSA) would be best represented in terms of both absolute end-RFU as well as the same after normalization against a standard. The former was expected to provide an absolute scale of NSA while the latter would represent a relative one. Rolling circle amplification (RCA), however, when recorded in a standard real-time PCR (rt-PCR) instrument, usually did not automatically generate either absolute or relative RFU. The proprietary software for the rt-PCR instrument, intuitively programmed to sense quantitative real-time PCR (qPCR) assays, presumably searches for an early flat baseline in an amplification curve. It then "subtracts" the baseline from the whole amplification to produce the characteristic and familiar "S"shaped plots with initial linear baselines. However, an RCA assay, in contrast to qPCR assays, may not have an early flat baseline. Rather, its fluorescence rises early in the amplification and generates a flat saturation plateau later in the assay. Due to the propensity to sense a flat baseline by the proprietary algorithm, the resulting baseline subtraction led to an inverted amplification plot with negative RFU (Figure and). To resolve this issue, we first manually tried to set the baseline identification at 1 st two cycles (cycles were set at 2 min with each having fluorescence monitoring throughout the 120 min assay time). This has corrected the negative RFU and inverted amplification plot issue (Figures and). However, this was still a misrepresentation as HRCA amplification RFU was marginally higher than that of LRCA. This misrepresentation was resolved when we removed all baseline subtraction from the amplification profiles, i.e., presented it as absolute RFU (Figure ). HRCA amplification RFU started increasing very early in the amplification and sometimes was 1.5 -2-fold higher compared to LRCA by the 2 nd cycle itself (Figure ). An early 1 -2 cycle baseline subtraction (as in Figures and) decreased the overall HRCA RFU and caused the stated misrepresentation (i.e., almost identical amplification profiles for LRCA and HRCA).