comprehensive high-throughput FTIR spectroscopy-based method for evaluating the transfection event: estimating the transfection efficiency and extracting associated metabolic responses

Reporter genes are routinely used in every laboratory for molecular and cellular biology for studying heterologous gene expression and general cellular biological mechanisms, such as transfection processes. Although well characterized and broadly implemented, reporter genes present serious limitatio...

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Bibliographic Details
Published in:Analytical and bioanalytical chemistry 2015-10, Vol.407 (26), p.8097-8108
Main Authors: Rosa, Filipa, Sales, Kevin C, Cunha, Bernardo R, Couto, Andreia, Lopes, Marta B, Calado, Cecília R. C
Format: Article
Language:English
Subjects:
adverse effects
Analytical Chemistry
Biochemistry
Biomarkers
Biotechnology
cell biology
Cell cycle
Cell lines
Cellular
Cellular biology
Characterization and Evaluation of Materials
chemical composition
Chemistry
Chemistry and Materials Science
Efficiency
Food Science
Fourier transform infrared spectroscopy
Fourier transforms
Gene expression
Genes
Genes, Reporter
Genetic aspects
HEK293 Cells
heterologous gene expression
High-Throughput Screening Assays - methods
Humans
Laboratory Medicine
least squares
Least-Squares Analysis
Metabolism
Metabolites
Metabolome
Methods
Monitoring/Environmental Analysis
Observations
Plasmids
Proteins
reporter genes
Research Paper
Spectra
spectral analysis
Spectroscopy
Spectroscopy, Fourier Transform Infrared - methods
Spectrum analysis
Transfection
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Summary:Reporter genes are routinely used in every laboratory for molecular and cellular biology for studying heterologous gene expression and general cellular biological mechanisms, such as transfection processes. Although well characterized and broadly implemented, reporter genes present serious limitations, either by involving time-consuming procedures or by presenting possible side effects on the expression of the heterologous gene or even in the general cellular metabolism. Fourier transform mid-infrared (FT-MIR) spectroscopy was evaluated to simultaneously analyze in a rapid (minutes) and high-throughput mode (using 96-wells microplates), the transfection efficiency, and the effect of the transfection process on the host cell biochemical composition and metabolism. Semi-adherent HEK and adherent AGS cell lines, transfected with the plasmid pVAX-GFP using Lipofectamine, were used as model systems. Good partial least squares (PLS) models were built to estimate the transfection efficiency, either considering each cell line independently (R ² ≥ 0.92; RMSECV ≤ 2 %) or simultaneously considering both cell lines (R ² = 0.90; RMSECV = 2 %). Additionally, the effect of the transfection process on the HEK cell biochemical and metabolic features could be evaluated directly from the FT-IR spectra. Due to the high sensitivity of the technique, it was also possible to discriminate the effect of the transfection process from the transfection reagent on KEK cells, e.g., by the analysis of spectral biomarkers and biochemical and metabolic features. The present results are far beyond what any reporter gene assay or other specific probe can offer for these purposes.
ISSN:1618-2642
1618-2650
DOI:10.1007/s00216-015-8983-9