Another important finding of our work is only splint-length 16 nt could lead to less than 5% non-specific amplification and a flat baseline under no target control for both LRCA and HRCA studies. However, we could not find any prior literature reports with 19 nt or lower splint length in the stated period. The observations from our study repeatedly underscored the high thermodynamic stability of the splint-cDNA complex that persisted even in denaturing PAGE. This observation indicated that the stability of splint-cDNA complex in denaturing PAGE for long splint length may still cause NSA even after gel purification of cDNA.