Signaltobackground and signaltonoise

Most microarray processing steps suffer from introducing high unspecific background by contamination or sub-optimal stringency of incubation and washing. The detector itself produces a dark current signal as a more or less flat (dark current noise) baseline background. This background can be subtracted by default from the image data by a given offset implemented by the system. The offset of a detection system cannot be simply measured by a user. Spreading the entire signal range over 16 bit, some holes in the histogram indicate the value of subtracting the dark current count. More substantially the background and the noise of the image can be an indicator of the image (and detection) quality. The 'quick and easy' evaluation is to take a reference slide with a bright (not saturated) signal and the background from a surrounding area (10 pixel distance to signal) and calculate the CVs and the signal-to-background ratio. For measuring the detection limit Tecan recommends to achieve a factor of 2 from SNR calculation ((raw intensity - background)/standard deviation background)). SNR can vary and are highly dependent upon the type of sample and what dyes are used. Although Tecan has not established specific SNR numbers for Cy3/Cy5 'standard' arrays, note that noise from the sample (vs noise from the instrument) limits SNR in 98% of cases. As a rule of thumb one could say that at maximum signal the LS Reloaded can achieve SNR of 5 logs. The electronic dark current count noise of LS Reloaded - independent of a sample - is typically 3-5 counts. Comparing two scanner systems it is recommended to use a set of two replicate slides on both systems and make sure that bleaching and degradation of the dyes is a non-issue for the test. Calibration or validation of fluorescence scanners is not trivial because most of the current test tools are not stable.

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