Computational protocol: The relationship between nuclear factor (NF)-κB family gene expression and prognosis in triple-negative breast cancer (TNBC) patients receiving adjuvant doxorubicin treatment

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Protocol publication

[…] The NanoString nCounter Analysis System (NanoString Technologies, Seattle, WA, USA) was used to measure the amount of gene expression. Using a multiplexed hybridization assay and digital readouts of fluorescent probes, this system measures the relative abundance of each mRNA transcript. We used an nCounter CodeSet (NanoString Technologies) containing biotinylated capture probes for NFKB1, NFKB2, RELA, RELB, REL, TP53, FOXC1, TBP, SP1, STAT3 and IRF1 genes and 5 housekeeping genes and reporter probes attached to color barcode tags according to the nCounter code-set design. The CodeSet was hybridized in solution to 200 ng of total RNA for 18 h at 65 °C according to the manufacturer’s instructions.Hybridized samples were loaded into the nCounter Prep Station for post hybridization processing. On the deck of the Prep Station, hybridized samples were purified and immobilized in a sample cartridge for data collection, followed by quantification of target mRNA in each sample using the nCounter Digital Analyzer. Quantified expression data were analyzed using NanoString’s nSolver Analysis Software.After performing image quality control using a predefined cutoff value, we excluded outlier samples using a normalization factor based on the sum of positive control counts greater than threefold. Counts of the probes were then normalized using the geometric mean of the five housekeeping genes and log2 transformed for further analysis. [...] DRFS was defined as the elapsed time from the date of curative surgery to the detection of disease recurrence. DRFS was analyzed using the Kaplan-Meier method. Univariate and multivariate analyses of DRFS were performed using Cox’s proportional hazards regression tests. To evaluate the relationship among expression of the eleven genes, we use Pearson correlation analysis. Lastly, ROC analysis was performed to evaluate the prognostic value of the expression level of NF-kB family genes and ligands adding weight. ROC analysis was conducted by adding the weighted value of gene expression on a pre-existing known prognostic marker already validated using univariate analysis. Two-tailed P values of <0.05 were considered statistically significant and IBM SPSS Statistics 21 for Windows (IBM Corp., Armonk, NY, USA) was used to analyze all data. […]

Pipeline specifications

Software tools nSolver Analysis Software, SPSS
Applications nCounter System, Miscellaneous
Organisms Homo sapiens
Diseases Breast Neoplasms, Neoplasms
Chemicals Doxorubicin