Next, we investigated various dual (i.e., in presence of both Exo I and III enzymes) digestion conditions having different buffer and salt compositions (conditions C5 -C9, Table ) for selfannealing ligated cDNA 1C, splint-padlock ligated 4C, and cohesive end ligated 11C. Such dual enzymatic digestions in the literature survey have predominantly been carried out without specific mention of buffer conditions (Figure ). However, preliminary data for dual digestions such as in C3 (ligase buffer condition) above indicated they would likely be more effective in mitigating NSA than single enzyme digestions. To investigate the role of buffers and salt conditions in probing NSA, we conducted pilot studies using cDNAs 1C, 4C, and 11C alone (Figure and) and compared the same with primer assisted LRCA (Figure and). In our pilot studies, interestingly, dual digestion in the Exo III buffer alone performed better than the presence of combined Exo I and Exo III buffers. This was not very well visible in raw amplification plots for the NSA of self-annealing ligated cDNA 1C as the RFU was already quite low (Figure ). But the effectiveness of dual digestion in Exo III buffer in 1X concentration over combined Exo I and III buffers in 1X concentration was prominently visible for both splint-padlock and cohesive end ligated cDNAs 4C and 11C (Figure and).