Figure 2

Ability of StaRT PCR to detect minute variations in transcript amounts. NT/CT ratios were designed to test the ability of StaRT PCR to detect minute changes in transcript quantity. cDNAs from LD419 and T24 cells were normalized using ACTB primers. The starting cDNA concentrations used for this experiment were different for T24 (equivalence point) and LD419 (approximately 1.5 × equivalence point). NT volumes decreasing successively by 0.4 μL were mixed with a constant volume of CT (4 μL of 10–12 M ACTB) to obtain NT/CT ratios over a range of 1.5:1 to 0.5:1. This allowed us to test the sensitivity of StaRT PCR over a range of 7%–15%. These reaction mixes were then subjected to StaRT PCR amplification using ACTB primers and the corresponding number of ACTB transcripts in the NT was calculated. The absolute number of ACTB transcripts quantified at each NT/CT ratio was plotted against the NT/CT ratio. StaRT PCR was sensitive enough to detect even the least tested change (7%) in the transcript amount (p < 0.01 using the paired Student's t-test).