Transient plasmid transfection is a common approach in studies in cultured mammalian cells. To examine behavior of transfected plasmids, we analyzed their transcriptional landscape by deep sequencing. We have found that the entire plasmid sequence is transcribed at different levels. Spurious transcription may have undesirable effects as some plasmids, when co-transfected, inhibited expression of luciferase reporters in a dose-dependent manner. In one case, we attributed this effect to a Kan/Neo resistance cassette, which generated a unique population of edited sense and antisense small RNAs. The unexpected complexity of expression from transiently transfected plasmids underscores the importance of appropriate experimental controls.

Institute of Molecular Genetics AS CR Prague Czech Republic

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

Cell-Based Reporter System for High-Throughput Screening of MicroRNA Pathway Inhibitors and Its Limitations

CRE promoter sites modulate alternative splicing via p300-mediated histone acetylation

Reporters transiently transfected into mammalian cells are highly sensitive to translational repression induced by dsRNA expression