Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids

Jana Nejepinska,Petr Svoboda,Radek Malik,Martin Moravec,Thomas Preiss

doi:10.1371/journal.pone.0043283

Jana Nejepinska, Petr Svoboda + Show 3 more

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https://doi.org/10.1371/journal.pone.0043283

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Abstract

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.

Highlights

Transient plasmid transfection is a routine approach to study gene expression in mammalian cells
We show here that 1) transfected plasmids are a rich source of spurious RNA transcription, 2) pEGFP and its derivatives carrying the same Kan/Neo backbone have strong negative effects on luciferase activity when compared to pBluescript, and 3) the most likely cause of luciferase reporter inhibition is double-stranded RNA (dsRNA) originating from co-transfected plasmids
Deep Sequencing Reveals Complex Expression of Transfected Plasmids phRL-SV40, pGL4-SV40, pEGFP-C1, and pBS plasmids were selected for deep sequencing. phRL-SV40 and pGL4-SV40 represent common Renilla and firefly luciferase reporter plasmids. pGL4-SV40 represents a newer generation of firefly luciferase reporters where putative mammalian transcription factor-binding sites in the plasmid backbone have been extensively mutated to minimize spurious expression [10]. pEGFP-C1 belongs to a family of plasmids for expressing protein fusions with the enhanced green fluorescent protein (EGFP). pBS is a common small cloning plasmid without any annotated eukaryotic transcription unit

Summary

Introduction

Transient plasmid transfection is a routine approach to study gene expression in mammalian cells. Ancestors of currently used reporter plasmids were developed one or two decades ago (firefly luciferase –1987 [1], Renilla luciferase – 1996 [2], green fluorescent protein –1994 [3]) when available technologies limited detailed analysis of plasmid expression and its behavior in transfected cells. The majority of common plasmids used in laboratories are commercial plasmids or their derivatives. Information about these plasmids is often restricted into company’s technical notes [10,11]) while peer-reviewed information about potential problems with these plasmids is limited or even absent Information about these plasmids is often restricted into company’s technical notes (e.g. [10,11]) while peer-reviewed information about potential problems with these plasmids is limited or even absent

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