Determination of linearity of NT/CT ratios over PCR cycles from 23–35: LD419 cDNA was mixed with the CT to arrive at NT/CT ratios: 0.1, 0.5, 1, 5, and 10. Independently, each of these 5 initial NT/CT ratios were used to set up separate reactions, which were then subjected to PCR amplification with ACTB primers and terminated at cycles 23, 26, 29, 32, 35. All experiments were performed in triplicate. The mean observed NT/CT ratios (following quantification using Bio-Rad Quantity One software) at each end point were plotted against the cycle numbers at which the reactions were terminated. The observed NT/CT ratios remain significantly constant during the StaRT PCR amplification cycle when the assigned NT/CT ratio is close to 1.