In the DASL assay, total RNA was first converted into cDNA in a reverse transcription reaction using biotinylated primers and resulted in fluorescent-labeled PCR products that were annealed to the BeadChips. The fluorescence of hybridized transcripts was captured by laser confocal microscopy scanning considering using the Illumina BeadArray Reader system [20]. The DASL assay was performed by the Fred Hutchinson Cancer Research Center (FHCRC) Genomics Resource. 2.5. Data Analysis The raw DASL array data was exported via genomeStudio version 1.0 (Illumina, Inc.) to JMP Genomics (SAS Institute, Inc., Cary, NC, USA) for quantile normalization, principal component analysis (PCA), and log transformation. The data analysis QC standard was performed on the sample and chip level to ensure uniformly high quality microarray data.
Initially, the quality of each BeadArray was assessed by examining the percent present calls (defined as the percent of bead types having a detection call P value <0.05), as well as plots of signal intensities for housekeeping, cy3_hyb, low_stringency_hyb, labeling, and biotin control bead types. In addition, log 2 transformation was performed, followed by quantile normalization and variance stabilizing transformation. For differential expression analyses, we followed the quintile with the greatest variance as previously described by Mittempergher and colleagues [21]. Genes differentially expressed between HCC-R and HCC-NR tumors were selected based on significance criteria of a false discovery rate less than 0.05 using analysis of variance (ANOVA) (��0.
05, with a fold change of 2). Heat maps were generated based on gene expression patterns through k-means clustering and viewed using CLUSTER 3.0 and TreeView 1.45 (software at http://eisenlab.org/), respectively. The threshold applied to our statistical tests was influenced by the large patient-to-patient variability in gene expression, although heterogeneity in expression is expected in human samples. Survival analysis was conducted using Kaplan-Meier curves and compared using the log-rank test. 2.6. Functional Data Analysis Network analysis of the differentially expressed genes was performed using Ingenuity Pathway Analysis software (IPA) (Ingenuity Systems, Inc., Redwood City, CA, USA). The top canonical pathways that represent differentially regulated genes in the tumor tissues from the HCC recurrence group were evaluated.
The top-scoring GSK-3 network of interactions is presented for the concurrent downregulated and the concurrent upregulated gene sets. This software analyzes molecular data in the context of known biological response and regulatory networks as well as other higher-order response pathways. Ingenuity functional analysis identified biological functions and/or diseases that were most significantly enriched and generated P values to determine the probability that each biological function assigned to that data set was due to chance alone. 2.7.
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