Analysis of the Level of Plasmid-Derived mRNA in the Presence of Residual Plasmid DNA by Two-Step Quantitative RT-PCR
Open Access
- 22 May 2020
- journal article
- research article
- Published by MDPI AG in Methods and Protocols
- Vol. 3 (2), 40
- https://doi.org/10.3390/mps3020040
Abstract
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.Keywords
This publication has 33 references indexed in Scilit:
- Simple statistical identification and removal of contaminant sequences in marker-gene and metagenomics dataMicrobiome, 2018
- Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNAJournal of Visualized Experiments, 2018
- A PCR-based approach to assess genomic DNA contamination in RNA: Application to rat RNA samplesAnalytical Biochemistry, 2016
- Pseudogenes as Weaknesses of ACTB (Actb) and GAPDH (Gapdh) Used as Reference Genes in Reverse Transcription and Polymerase Chain ReactionsPLOS ONE, 2012
- Correction of RT–qPCR data for genomic DNA-derived signals with ValidPrimeNucleic Acids Research, 2012
- mRNA-specific reverse transcription-polymerase chain reaction from human tissue extractsAnalytical Biochemistry, 2002
- PEX11β Deficiency Is Lethal and Impairs Neuronal Migration but Does Not Abrogate Peroxisome FunctionMolecular and Cellular Biology, 2002
- Exclusive Amplification of cDNA Template (EXACT) RT-PCR to Avoid Amplifying Contaminating GenomicPseudogenesBioTechniques, 2001
- RNA template-specific polymerase chain reaction (RS-PCR): a novel strategy to reduce dramatically false positivesGene, 1990
- In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria.The Journal of cell biology, 1983