Microarray analyses were applied to tumor and metastasis biopsies from 13 individuals following a randomized and blinded unbalanced design. Additional human normal colon samples (CA, CB, CC), one colon adenocarcinoma sample (CT) and one normal liver (CL) tissue sample (Stratagene) were included as calibration controls but were not considered in statistical analyses. Uneven numbers of samples were randomly allocated to each of the engineers who were not aware of sample phenotypes. To assess data reproducibility and minimize dye bias effects, each of the samples was measured four times, twice with Cy3 and twice with Cy5. To ensure robustness and flexibility in data analysis, a reference design was used with a universal reference sample (Stratagene) serving as a baseline for the comparisons of tumor samples. Such a design does not require pre-definition of the subgroups for comparison, allows robust discovery of non-anticipated classes among the samples and is compatible with subsequent additional sampling.
Statistical power (1-β) for t statistics of the experimental design was computed for estimation of false negatives (FNR) and FDRs [99 (link)] as shown in Additional data file 3. This calculation is based on the observation that the gene-specific expression measurements are approximately normally distributed, and takes into account the accepted confidence level (α), the magnitude of the effect measured (Φ), the biological variation (σ) expected in the population investigated, and the size of the groups of samples from individual patients (n1, n2). Statistical comparison was done considering that biopsies collected prior to drug exposure may be subsequently categorized in two groups of chemo-sensitive (complete and partial responses) or resistant (progressive and stable diseases; Additional data file 10) samples in view of the initial response rates of individual patients to combined chemotherapy [11 (link)].
Statistical power (1-β) for t statistics of the experimental design was computed for estimation of false negatives (FNR) and FDRs [99 (link)] as shown in Additional data file 3. This calculation is based on the observation that the gene-specific expression measurements are approximately normally distributed, and takes into account the accepted confidence level (α), the magnitude of the effect measured (Φ), the biological variation (σ) expected in the population investigated, and the size of the groups of samples from individual patients (n1, n2). Statistical comparison was done considering that biopsies collected prior to drug exposure may be subsequently categorized in two groups of chemo-sensitive (complete and partial responses) or resistant (progressive and stable diseases; Additional data file 10) samples in view of the initial response rates of individual patients to combined chemotherapy [11 (link)].
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