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Further scaling the data to a common reference sample (herein referred to as total count scaling) introduces more variability by pushing all samples toward the same distribution, especially for samples with different sequencing depth.
For all the FF tumors and the Common Reference Sample, Agilent 244,000 feature whole genome microarrays were hybridized with tumor RNAs (Cy5) and a human common reference (Cy3) and lowess normalized as described in Herschkowitz et al. [ 30].
A common reference sample was constructed from a pool of all labeled RNA extracts.
The same common reference sample was thus used in all the 46 (2×23) hybridizations.
Allele size lengths have been standardized via comparison with a common reference sample.
Data are expressed as log2 ratios of fluorescence intensities of the experimental and the common reference sample.
In each measurement, individual samples were measured together with a common reference sample comprised of all 12 samples used in this study.
As cluster analysis was the major goal of this study the microarray experiment followed a reference design, where all samples were hybridized against a common reference sample.
One labeled tissue sample RNA (Alexa 594) and one labeled common reference sample RNA (Alexa 488) was thus hybridized to each of 46 microarrays.
Expression ratios were defined by the signal intensity of channel one (tissue sample), divided by the signal intensity of channel two (common reference sample).
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The log2 ratio of RNA-Seq tumor samples to RNA-Seq human Common Reference Sample (which was the same RNA used for the 2-color microarrays) was determined.
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