Deregulation of miRNAs in FTO knockdown cells.

Mature miRNAs showing increased (A) and decreased (B) steady state levels in FTO knockdown cells. Normalized RNA-seq read numbers of individual miRNAs in FTO knockdown and scrambled siRNA treated cells were compared. Mean ± SD of three independent experiments are depicted. For verification and further studies, qRT-PCR analyses of selected mature miRNAs (C) and primary miRNA transcripts (D) were performed. We did not use other small RNAs as a reference gene for measuring mature miRNAs levels (as suggested by Life Technologies), since depletion of FTO might have an impact on their levels. Therefore, luciferase RNA was used to generate a standard curve and added to the qRT-PCR assays. GAPDH was used as a reference gene for measuring primary miRNAs transcript levels. Mean ± SD from quadruplicates per assay for three independent cell lines (FTO1C1, FTO2D4 and FTO3C3) are depicted. kd, FTO specific siRNA treated cells, scr, scrambled siRNA treated cells.