Figure 2: The end point fluorescence of the ~20 pl microwell PCR reaction is proportional to the logarithm of the initial amount of target DNA (that is, log-linear).

(a) A representative image of a 15,000-well array before PCR. Scale bar, 40 μm. (b) The clear log-linear trend between well intensity at cycle 40, and the number of cells per well after amplifying the promoter region of the Nanog gene suggests that the well intensity is a semi-quantitative indicator of the initial amount of target DNA. Error bars represent s.e. (n=50 for 0–3 cells per well and n=4 for 4 cells per well). Note that the initial background fluorescence coming from the nuclear DNA before PCR has been subtracted, and the mean intensity is the signal from the free in-solution fluorescence. (c) Representative image from the microwell array after 40 cycles for PCR. Scale bar, 40 μm. (d) Magnified image of a representative region with wells containing, 0, 1, 2, 3 and 4 cells. The array was loaded with ~500 cell per μl in a total volume of 60 μl. The dashed lines represent the well boundaries. Note that all pixels that correspond to the fluorescent intensity from the cells are discarded from the quantification, thus only the area corresponding to the free, in-solution fluorescence is quantified. Scale bar, 20 μm. (e) Log-linear relation between end point fluorescence (40 cycles) and starting number of spiked in pAW109 RNA templates. Error bars represent s.e. (n=20).