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The numbers of clusters in the GMM for the SP, NSP, and background were set at 5, 10, and 2, respectively.
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Background was set at 3 standard deviations above the average A405 readings of 3 wells to which no primary antibody was added.
The significance level for tests related to patient background was set at 15% for both sides.
Average tissue background was set at the minimum on a gray scale of 255 shades.
The experimental background was set at around 7.5 and an intensity of less than 9 corresponded to low expression.
The resulting expression levels for each of the miRNAs measured by the microarrays were calculated and filtered for miRNAs expressed above a background level (background was set at 30, approximating the median signal per array).
Arbitrarily, the intensity of the region prior to bleaching was set at 1 while that of the background was set at 0. Relative intensities are represented as a function of time.
For MLR to allogeneic cells, CD4+ T cells isolated from mice of C57BL/6 background were set up at 2 × 10 cells/well and cocultured for 3 days with 2 × 10 cells/well of mitomycin C-treated BALB/c allogeneic splenocytes.
Background gates were set at 0.05%, and positive results were defined as >2-fold >2-foldund.
In the second step, elements that had intensities below twice the background intensity in one channel only were set at twice background levels.
In brief, image expression data was used as channel intensity minus background and intensity thresholds were set at a value of 300.
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