1. Select a slide that is representative of your typical data slides.
2. Scan the slides at a PMT setting that gives the brightest image without any saturating pixels.
3. In Excel, divide the S-B value (e.g., the F532 Median-B532 column) by the N value (e.g., the B532 SD column).
The type of PMT used in the GenePix 4000 Array Scanners has a wide dynamic range, so it detects and amplifies both strong specific signal on arrayed spots, and low-level background fluorescence, which is primarily due to non-specific hybridization. Simply increasing the gain to the maximum setting may not produce the optimum SNR. Thus, the optimal PMT setting must be determined empirically. The first step is to make a dilution series of the probes to be used. Next, the relationship between the SNR and PMT gain is determined. From these data the optimal PMT settings for the fluorescent probes is obtained. Data presented below illustrates the relationship between a wide range of PMT voltages and the concentration for the commonly used Cy3 and Cy5 probes.
1. SNR as a Function of PMT Gain for a Single Dye Concentration.
The following experiments indicate that the signal-to-noise ratio continues to improve up to about 500 V. PMT gain above 500 V will give equal SNR for dye concentrations at the middle of the concentration range (Figure 1A), as well as near the detection limit (Figure 1B).
Figure 1: SNR for both the Cy5 (Red line) and Cy3 (Green line) was measured