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Analysis of the Level of Plasmid-Derived mRNA in the Presence of Residual Plasmid DNA by Two-Step Quantitative RT-PCR

Institute for Anatomy and Cell Biology, Division of Medical Cell Biology, Justus Liebig University, Aulweg 123, 35385 Giessen, Germany
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Methods Protoc. 2020, 3(2), 40; https://doi.org/10.3390/mps3020040
Received: 19 February 2020 / Revised: 19 May 2020 / Accepted: 21 May 2020 / Published: 23 May 2020
In transfection experiments with mammalian cells aiming to overexpress a specific protein, it is often necessary to correctly quantify the level of the recombinant and the corresponding endogenous mRNA. In our case, mouse calvarial osteoblasts were transfected with a vector containing the complete Pex11β cDNA (plasmid DNA). The Pex11β mRNA level, as calculated using the RT-qPCR product, was unrealistically higher (>1000-fold) in transfected compared to non-transfected cells, and we assumed that there were large amounts of contaminating plasmid DNA in the RNA sample. Thus, we searched for a simple way to distinguish between plasmid-derived mRNA, endogenous genome-derived mRNA and plasmid DNA, with minimal changes to standard RT-PCR techniques. We succeeded by performing a plasmid mRNA-specific reverse transcription, and the plasmid cDNA was additionally tagged with a nonsense tail. A subsequent standard qPCR was conducted using appropriate PCR primers annealing to the plasmid cDNA and to the nonsense tail. Using this method, we were able to determine the specific amount of mRNA derived from the transfected plasmid DNA in comparison to the endogenous genome-derived mRNA, and thus the transfection and transcription efficiency. View Full-Text
Keywords: endogenous genome-derived mRNA; genomic DNA; DNA contamination; nonsense-tail reverse transcription; nonsense-tail PCR primer; overexpression experiments; plasmid DNA; plasmid-derived mRNA endogenous genome-derived mRNA; genomic DNA; DNA contamination; nonsense-tail reverse transcription; nonsense-tail PCR primer; overexpression experiments; plasmid DNA; plasmid-derived mRNA
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MDPI and ACS Style

Ahlemeyer, B.; Colasante, C.; Baumgart-Vogt, E. Analysis of the Level of Plasmid-Derived mRNA in the Presence of Residual Plasmid DNA by Two-Step Quantitative RT-PCR. Methods Protoc. 2020, 3, 40. https://doi.org/10.3390/mps3020040

AMA Style

Ahlemeyer B, Colasante C, Baumgart-Vogt E. Analysis of the Level of Plasmid-Derived mRNA in the Presence of Residual Plasmid DNA by Two-Step Quantitative RT-PCR. Methods and Protocols. 2020; 3(2):40. https://doi.org/10.3390/mps3020040

Chicago/Turabian Style

Ahlemeyer, Barbara, Claudia Colasante, and Eveline Baumgart-Vogt. 2020. "Analysis of the Level of Plasmid-Derived mRNA in the Presence of Residual Plasmid DNA by Two-Step Quantitative RT-PCR" Methods and Protocols 3, no. 2: 40. https://doi.org/10.3390/mps3020040

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