Validating RNA-seq Differential Expression

16 RNA samples were obtained by the procedures, described above, from another batch of female Brd1^+/− and WT mice (8/group). 180 ng total RNA was reverse transcribed by iScript Select cDNA Synthesis Kit (Bio-Rad, Hercules, USA). All eight DEGs, detected by DESeq2 and TSPM, were selected for validation. 107 more genes were randomly selected for validation from the list of remaining 191 DEGs, detected by Cuffdiff2 and edgeR. After 10–20 cycles of specific target amplification with PreAmp master mix (Fluidigm, San Francisco, USA), high-throughput qPCR was performed on the BioMark HD (Fluidigm, San Francisco, USA), using 48.48 dynamic arrays (Fluidigm, San Francisco, USA) and SsoFast EvaGreen Low ROX kit (Bio-Rad, Hercules, USA) [Additional files 12 & 13]. A DEG, detected by the RNA-seq DEG analysis methods, was considered as a true-positive DEG, if it satisfied the following criteria, (i) Both RNA-seq and qPCR showed same direction (upregulation or down-regulation) of differential expression, (ii) Differential expression fold change, estimated by qPCR, was either above 1.25 or below 0.80 (LFC cut-off was ±0.3219) [34 (link)]. Spearman correlation coefficients, root-mean-square deviations and kappa statistics were calculated using STATA 13.1 (StataCorp LP, Texas, USA).