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

Barbara Ahlemeyer,Eveline Baumgart-Vogt,Claudia Colasante

doi: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.

Highlights

Reverse transcriptase quantitative PCR (RT-qPCR)-based gene expression analysis of cells transfected with plasmid DNA requires the isolated RNA to be free from contaminating genomic and plasmid DNA
An oligo(dT) RT primer tagged with a unique sequence [6] can be used for reverse transcription, and the detection of the tagged cDNA during qPCR is conducted by using a reverse PCR primer matching the unique sequence
We succeeded by modifying the primer design suggested by Smith et al [8] and produced an RT primer, in which the first part annealed to the 3 ́-end of the plasmid-derived mRNA, followed by a poly(T)17 tail (which annealed to the poly(A)17 of the plasmid-derived mRNA) and, a nonsense nucleotide sequence

Summary

Introduction

Reverse transcriptase quantitative PCR (RT-qPCR)-based gene expression analysis of cells transfected with plasmid DNA requires the isolated RNA to be free from contaminating genomic and plasmid DNA. An oligo(dT) RT primer tagged with a unique sequence [6] can be used for reverse transcription, and the detection of the tagged cDNA during qPCR is conducted by using a reverse PCR primer matching the unique sequence Both ways, will not work in the presence of processed pseudogenes or high amounts of residual plasmid DNA (e.g., in transfection experiments), as these DNA species do not have introns and poly(A) tails. The qPCR was performed with PCR primers annealing to the 3 ́-end of the plasmid cDNA (forward primer) and to the nonsense sequence tag (reverse primer), which was incorporated into the plasmid cDNA during the RT-PCR This allowed for the selective amplification of plasmid-derived cDNA and the quantification of the plasmid-derived mRNA level, even in the presence of contaminating plasmid DNA

Materials

Transfection of the MC3T3-E1 Cell Line

Detection of Contaminating Plasmid DNA in Transfected MC3T3-E1 cells

Necessity for a Selective Detection of Plasmid cDNA