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(a, b) Background histograms of two patients, (c) background standard deviation for all patients, and (d) background average plus three times its standard deviation.
The noise properties of the data can be appreciated in Figure 3, which illustrates the background histograms of two patients and the resulting background standard deviation for all patients.
The data was filtered using a signal to noise ratio criteria and only features for which the background-subtracted signal was 2.6 times above the background standard deviation for that feature in at least 13 of the 15 arrays, were retained.
Likewise, a more rigorous threshold was applied by using the flag IsWellAboveBG, which first determines if the feature is significant (IsPosAndSignif) and then determines if the background-subtracted signal is greater than 2.6 times the background standard deviation for that feature (approximates a 99% CI).
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Finally, the average of all pixels above a threshold value of three times the background standard deviation was calculated for the three slices for each cell for Figure 5.
The signal-to-noise ratio [SNR; SNR = (signal mean - background mean)/(background standard deviation)] was then calculated for each spot to discriminate true signals from noise.
The Genepix result files, including signal, background, standard deviation, pixel statistics and quality parameters for both channels were deposited in the microarray database (mAdb) maintained by the NCI/CIT bioinformatics group [ 57].
We quantitated the scanned images with ScanArray Express 2.0 using the adaptive thresholding quantitation algorithm to generate values for median signal intensity, median background intensity, and background intensity standard deviation for each spot on the array.
Here, unusable may mean: i) a bad quality flag or ii) a gene expression value that is lower than the average background + 2 times its standard deviation for both samples measured on a given spot.
The quantification cycle (Cq) values for each reaction were calculated automatically by the 7300 Real-Time PCR system detection software by determining the point in time (PCR cycle number) at which the reporter fluorescence exceeded the computer-determined standard deviation for background by a factor of 10.
The arithmetic mean and standard deviation for the background corrected log transformed red and green intensities were 6.97, SD = 4.21 and 7.90, SD = 3.25, respectively.
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CEO of Professional Science Editing for Scientists @ prosciediting.com