Optimization of a quantitative PCR based method for plasmid copy number determination in human cell lines

Fliedl, Lukas; Kast, Florian; Grillari, Johannes; Wieser, Matthias; Grillari‐Voglauer, Regina

doi:10.1016/j.nbt.2015.03.004

Cited by 8 publications

(8 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This suggests that sufficient numbers of intact plasmids reach the cell nucleus to facilitate NK1 gene expression even at those ratios. Studies have shown that during a transfection event, individual cells can take up about 10 5 –10 6 copies of a plasmid [ 16 ]. However, as discussed earlier, plasmids also need to reach the nucleus in order to be expressed, but are prone to rapid degradation by nucleases naturally present in the cytoplasm [ 14 ].…”

Section: Discussionmentioning

confidence: 99%

“…These aspects have been studied for both surface-immobilized and solution-mediated transfection systems [ 10 , 11 , 12 ]. Other important parameters influencing protein expression efficiency are the cell cycle [ 13 , 14 ], the concentration [ 15 , 16 , 17 ] and the purity of plasmid DNA [ 18 ]. Higher plasmid concentrations have a direct effect on intracellular plasmid copy numbers in both the cytoplasm and the nucleus up to a certain maximum.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Calcium Imaging of GPCR Activation Using Arrays of Reverse Transfected HEK293 Cells in a Microfluidic System

Roelse¹,

Henquet²,

Verhoeven³

et al. 2018

Sensors

View full text Add to dashboard Cite

Reverse-transfected cell arrays in microfluidic systems have great potential to perform large-scale parallel screening of G protein-coupled receptor (GPCR) activation. Here, we report the preparation of a novel platform using reverse transfection of HEK293 cells, imaging by stereo-fluorescence microscopy in a flowcell format, real-time monitoring of cytosolic calcium ion fluctuations using the fluorescent protein Cameleon and analysis of GPCR responses to sequential sample exposures. To determine the relationship between DNA concentration and gene expression, we analyzed cell arrays made with variable concentrations of plasmid DNA encoding fluorescent proteins and the Neurokinin 1 (NK1) receptor. We observed pronounced effects on gene expression of both the specific and total DNA concentration. Reverse transfected spots with NK1 plasmid DNA at 1% of total DNA still resulted in detectable NK1 activation when exposed to its ligand. By varying the GPCR DNA concentration in reverse transfection, the sensitivity and robustness of the receptor response for sequential sample exposures was optimized. An injection series is shown for an array containing the NK1 receptor, bitter receptor TAS2R8 and controls. Both receptors were exposed 14 times to alternating samples of two ligands. Specific responses remained reproducible. This platform introduces new opportunities for high throughput screening of GPCR libraries.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Calcium Imaging of GPCR Activation Using Arrays of Reverse Transfected HEK293 Cells in a Microfluidic System

Roelse¹,

Henquet²,

Verhoeven³

et al. 2018

Sensors

View full text Add to dashboard Cite

show abstract

“…It is thought that CpG motifs present inside plasmids caused the stimulation of the immune system [25]. In addition, due to the limitation of the translation machinery, considerable amounts of plasmid-derived mRNA often remained untranslated [26,27].…”

Section: A Simple Rt-qpcr Protocol To Distinguish Between Plasmid-dermentioning

confidence: 99%

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

Ahlemeyer

Colasante

Baumgart-Vogt

2020

MPs

View full text Add to dashboard Cite

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.

show abstract

“…This suggests that sufficient numbers of intact plasmids reach the cell nucleus to facilitate NK1 gene expression even at those ratios. Studies have shown that during a transfection event, individual cells can take up about 10 5 to 10 6 copies of a plasmid [178]. However, as discussed earlier, plasmids also need to reach the nucleus in order to be expressed, but are prone to rapid degradation by nucleases naturally present in the cytoplasm [176].…”

Section: Discussionmentioning

confidence: 99%

“…These aspects have been studied for both surface-immobilized and solution-mediated transfection systems [172][173][174]. Other important parameters influencing protein expression efficiency are the cell cycle [175,176] the concentration [177][178][179], and the purity of plasmid DNA [180]. Higher plasmid concentrations have a direct effect on intracellular plasmid copy numbers in both the cytoplasm and the nucleus up to a certain maximum.…”

Section: Introductionmentioning

confidence: 99%

Receptomics, design of a microfluidic receptor screening technology

Roelse¹

View full text Add to dashboard Cite

Chapter 1 Introduction Chapter 2 A generic microfluidic biosensor of GPCR-activation-monitoring cytoplasmic Ca 2+ flux in human HEK293 cells Chapter 3 Metabolomics meets functional assays: coupling LC-MS and microfluidic cell-based receptor-ligand analyses Chapter 4 Calcium imaging of GPCR activation using arrays of reverse transfected HEK293 cells in a microfluidic system Chapter 5 The effect of calcium buffering and calcium sensor type on the sensitivity of an array-based bitter receptor screening assay Chapter 6 Statistical models discriminating between complex samples measured with microfluidic receptor-cell arrays Chapter 7 General discussion References Summary Nederlandse samenvatting Abbreviations Acknow ledgements About the author List of publications Overview of completed training activities C H A P T E R 18 Chapter 1 | Introduction Figure 3, Receptor cell assay platforms (diagram adapted from Martins et al 2012). A, The multiwell plate format; cells are seeded on the surface of a multi well plate and (top-down) transfected with receptor coding plasmid DNA. Calcium dye loading, subsequent washing steps and test sample dosing are done using fluid dispensers. The test conditions are multiplexed by using multiple wells and multiple plates making this platform compatible with automation but at relatively high costs and low assay flexibility [90]. B, Receptor cell arrays are created within one well chamber by printing cells [97] or by printing receptor coding plasmid DNA into a micro well and by reverse transfection with cells [81, 85]. Arrays are co-transfected with fluorescent indicator proteins to locate the array. Array washing and sample dosing are done using fluid dispensers. This combines the HTS nature of multi-well plates with the possibility to screen multiple receptors and controls in a single well. C, In microfluidic platforms the cells are cultured or assembled into a microfluidic system [98, 99]. A system can be designed so that different channels address different chambers with (reverse transfected) cells analogous to A but with the option of repeated exposures [84] or a reverse transfected array can be enclosed in one large flowcell, sequentially addressing all spots at once with different samples analogous to B but with the option of repeated challenges and larger arrays [100]. Fluid dispenser Fluid dispenser Adherent cells Substrate Cover layer / flowcell with gasket Fluid Multiwell plate Adherent cell layer Adherent cells Substrate / well plate (A) Well plate HTS (B) Cell microarrays (C) Cell microfluidics Live cell assay platform Multiplexing Single flowcell cell array: Receptomics Multiple chambers Cell array in a well or on a slide Multiple well plates

show abstract

Optimization of a quantitative PCR based method for plasmid copy number determination in human cell lines

Cited by 8 publications

References 20 publications

Calcium Imaging of GPCR Activation Using Arrays of Reverse Transfected HEK293 Cells in a Microfluidic System

Calcium Imaging of GPCR Activation Using Arrays of Reverse Transfected HEK293 Cells in a Microfluidic System

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

Receptomics, design of a microfluidic receptor screening technology

Contact Info

Product

Resources

About