In order to validate the microarray results, gene specific primers were designed for 13 differentially expressed T. urticae genes (7 up- and 6 down-regulated genes) using Primer 3 v0.4.0.
To verify the microarray results, four genes of interest (2 up-regulated genes: vwbp, scpA and 2 down-regulated genes agrA, and hla) were subjected to qRT-PCR using the same RNA samples.
To validate the microarray results, fourteen genes were selected to include genes that were significantly upregulated (OCT4, CMYC, GATA4), downregulated (XIST, NANOG, GSTM3, CCNB1)) and non-significantly (VEGF, BCL2, SOX2, CDX2, HNF4a, BMPR1B, SMAD1) deregulated according to the microarray results.
To confirm the microarray results, nighteen genes involving in metabolism, information transfer, plasmid encoding genes and hypothetical proteins were chosen for the qPCR analysis (Table 2).
Within the ovary-upregulated category, we randomly chose two of the top 200 most upregulated genes, in order to assess the accuracy of microarray results for genes demonstrating less striking differences in expression.
To further highlight the clinical significance of the genes mapping to recurrent copy number altered regions in gastric cancers as well as to validate the microarray results, eleven genes were selected for the affinity capture based transcript analysis (TRAC assay).
To verify microarray results, five genes of interest were subjected to TaqMan real-time quantitative reverse-transcriptase PCR (Fig. 4B).
In order to confirm the microarray results, ten genes were chosen (five up-regulated and five down-regulated) for analysis using quantitative real time PCR (Q-PCR).
RT-qPCR analysis was able to recapitulate the microarray results of genes shown to be up- or downregulated in a H2B K37A mutant strain relative to the isogenic parent strain (Figure S1 and data not shown), thus validating the microarray results.
