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Deep sequencing reveals complex spurious transcription from transiently transfected plasmids.

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.

SUBMITTER: Nejepinska J

PROVIDER: S-EPMC3420890 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Deep sequencing reveals complex spurious transcription from transiently transfected plasmids.

Nejepinska Jana J Malik Radek R Moravec Martin M Svoboda Petr P

PloS one 20120816 8

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 ...[more]

PMID: 22916237

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Project description:Influenza A viruses frequently change their genetic characteristics, which leads to the emergence of new viruses. Consequently, elucidation of the relationship between influenza A virus and host cells has a great importance to cope with viral infections. In this study, it was aimed to determine expression profiles of interferon response genes in human embryonic kidney 293 (HEK293) cells infected with human (A/WSN-H1N1) and avian influenza A viruses (duck/Pennsylvania/10218/84/H5N2) or transfected with plasmids encoding viral RdRP subunits and, to obtain clues about the genes that may be important for the viral pathogenesis. The HEK293 cells cultured in a 12-well plate were infected with influenza A viruses or transfected with plasmids encoding viral polymerase. Total RNA extraction and cDNA preparation were carried out with commercial kits. Qiagen 96-well-RT2 Profiler PCR Array plates designated for interferons response genes were used for quantitation of the transcripts. The relative quantities of transcripts were normalized with STAT3 gen, and the results were evaluated. Quantitative RT-PCR results showed that there are substantial differences of the interferon response gene transcription in cells infected with viruses or transfected with plasmids. A higher number of interferon-related genes were found to be downregulated in the cells infected with DkPen compared to WSN. On the other hand, significant differences in the expression profiles of interferon response genes were observed in the cells expressing viral PA protein. In particular, avian influenza PA protein was found to cause more aggressive changes on the transcript levels. Human and avian influenza A viruses cause a substantial change in interferon response gene expression in HEK293 cells. However, a higher number of genes were downregulated in the cells infected with avian influenza DkPen compared to WSN. It has been also concluded that the viral PA protein is one of the important viral factors affecting the transcript level of host genes.

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Deep sequencing reveals complex spurious transcription from transiently transfected plasmids.

Publications

Deep sequencing reveals complex spurious transcription from transiently transfected plasmids.

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