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We will maintain the original analysis (using ACTIN measurements for normalization) and include an additional exploratory analysis from the original, using 36B4 to normalize the gene expression.
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We used cell-line controls to span the dynamic range of the measurements, allowing for normalization of the data across multiple batches and ensuring our ability to compare different batches.
U6 snRNA was measured for normalization.
The Renilla luciferase activity measurements were used for normalization.
In order to further compare the performances of different normalization and background correction methods, at each p-value cutoff point we calculate the average of each statistical measurement for each normalization method (across five different background correction methods) and for each background correction method (across four different normalization methods).
PCR efficiency was calculated from standard curves and the expression of 36b4 (time series measurements) or Gapdh (verification of microarray measurements, Figure 5) was used for normalization.
For normalization of measurements, photographic documentation was obtained as follows.
In the specific example of Fig. 2, top, one can distinguish the emission of S (from the surfactant) and of argon (from the gas in the measurement chamber) which may serve for normalization, and the emission of K+ and of Cs+.
In contrast, 800 nm and 805 nm were used for reference measurements and normalization of signals because they are in the middle of the typical range of 795 nm to 810 nm reported in the literature for the isosbestic point [ 15, 16, 21], where optical absorption of blood does not depend on oxygenation.
Therefore triplicate measurement of RPS28 mRNA was chosen for normalization of each sample.
Figure 2C illustrates the profound effects of the plasmid used for normalization in the measurement of the induction of NF-κB.
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