Figure 5

Probe-sets accurately reporting FOXM1 and MYB transcript levels. (a) mRNA levels for FOXM1 and MYB by qRT-PCR 24 h after lentiviral-mediated shRNA expression of a non-target sequence (NT; black bars); FOXM1- and MYB-specific sequences are shown by white bars. (b) FOXM1 and MYB protein levels by immunoblot 24 h after lentiviral-mediated shRNA expression of a non-target sequence (Control), and FOXM1- and MYB-specific sequences. (c) Affymetrix probe sets associated with FOXM1 and MYB transcripts fail to conclusively describe the effects of shRNA-mediated silencing. After FOXM1 and MYB silencing, some Affymetrix-annotated probe sets suggest mild increase in FOXM1 (two of three) and MYB expression (one of eight). Probe remapping to RefSeq transcripts and Cleaner annotation suggest that probes assigned to these probe sets either fail to match target genes or are inconsistent. As a result, Cleaner produced single probe clusters that correctly capture the shRNA-mediated knock-down validated by qRT-PCR.