Main constrains for RNAi induced by expressed long dsRNA in mouse cells

Using a system expressing dsRNA molecules and luciferase reporters, we explored factors contributing to RNAi inefficiency in mouse embryonic stem cells (ESCs) and NIH3T3 fibroblasts that could explain previous contradictions concerning mammalian RNAi. Low and poorly processive Dicer activity combined with insufficiently abundant substrates appear to be the primary reason for RNAi inefficiency. Bulk small RNA-seq of 8 NIH3T3 fibroblasts samples: - 2 replicates transfected with pCag-EGFP Lin28IR - 2 replicates transfected with pCag-EGFP MosIR - 2 replicates transfected with pU6 Lin28IR - 2 replicates transfected with pU6 MosIR