Under HRCA condition, self-annealing ligated cDNA 1C demonstrated the lowest NSA (end-RFU at 92.4 ± 47.1 and NNSA 5 ± 3.8) for the C5 condition, which was in turn 4-and 5-fold lower than that of C6 and C7 conditions, respectively (Figure and Figure ). This, the lowest possible end-RFU recorded for self-annealing ligation under HRCA condition in this study, led to an almost flat baseline in its representative amplification plot (Figure ). In a perplexing observation, however, the HRCA primer assisted NSA in cohesive end ligated cDNA 11C was very high for C5 -C7 conditions (80.2, 82.1, and 81.8% NNSA for conditions C5, C6, and C7, respectively) (Figure and Figure ). Overall, these data reflected successful mitigation of NSA for self-annealing ligation even under HRCA condition. But for cohesive end ligation, despite an almost absent NSA under LRCA conditions, conditions C5 -C7 interestingly demonstrated a high NSA under HRCA primer assisted amplification.