Figure 4
Verification of RNA-Seq and Agilent DEG identification by WaferGen SmartChip Real-Time PCR analysis. (A) QRTPCR data (n = 5) was used as the “gold standard” to determine true and false positives and negatives for RNA-Seq (n = 3) and Agilent (n = 3) datasets. (B) Performance metrics of RNA-Seq and Agilent validated by QRTPCR. (C) Representative example of a false-negative and (D) false-positive response in the RNA-Seq dataset. Official gene symbols are indicated in upper left corner with the number of RNA-Seq aligned reads in parentheses () and number of samples with Ct values lower than background in brackets [] for vehicle control samples. Bars represent mean fold-change determined by WaferGen technology (±SEM), the red line represents RNASeq fold-change, and the green line represents Agilent fold change. Significant differences within WaferGen data were determined by one-way ANOVA followed by Dunnett’s post-hoc test and indicated by an asterisk (*) with the exception of Fam83a whose undetectable levels prevented statistical testing. Red (RNA-Seq) and green (Agilent) dots represent P1 (t) values with size indicating level of significance (small ~0.8, large ~1). Labels on the X-axis indicate the dose of TCDD (μg/kg), number of aligned RNA-Seq reads, and number of samples with Ct values lower than background. Dashed lines indicate 1.5 and 2.0 |fold-change| thresholds to identify DEGs. (E) UCSC genome browser track illustrating PCR primer and Agilent probe alignments for Zfp846. The Zfp846 loci is presented in blue with exons (closed boxes) and introns (solid line). The arrowheads indicate the direction of transcription.