A comprehensive high-throughput FTIR spectroscopy-based method for evaluating the transfection event: estimating the transfection efficiency and extracting associated metabolic responses.
Anal Bioanal Chem
; 407(26): 8097-108, 2015 Oct.
Article
en En
| MEDLINE
| ID: mdl-26329279
ABSTRACT
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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Transfección
/
Espectroscopía Infrarroja por Transformada de Fourier
Tipo de estudio:
Risk_factors_studies
Límite:
Humans
Idioma:
En
Revista:
Anal Bioanal Chem
Año:
2015
Tipo del documento:
Article
País de afiliación:
Portugal