TY - JOUR
T1 - A comprehensive high-throughput FTIR spectroscopy-based method for evaluating the transfection event
T2 - estimating the transfection efficiency and extracting associated metabolic responses
AU - Rosa, Filipa
AU - Sales, Kevin C.
AU - Cunha, Bernardo R.
AU - Couto, Andreia
AU - Lopes, Marta B.
AU - Calado, Cecília R. C.
N1 - Funding Information:
The Innovation Agency (Portugal) is acknowledged through the financial support of the CLARO project. Marta B. Lopes gratefully acknowledges the post-doctoral scholarship from FCT (SFRH/BPD/73758/2010). The present work was partly conducted in the BioEngineering Laboratory resulting from the Protocol between Universidade Católica Portuguesa and the Instituto Politécnico de Lisboa.
Publisher Copyright:
© 2015 Springer-Verlag Berlin Heidelberg
PY - 2015/9/2
Y1 - 2015/9/2
N2 - 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 (R2 ≥ 0.92; RMSECV ≤ 2 %) or simultaneously considering both cell lines (R2 = 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.
AB - 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 (R2 ≥ 0.92; RMSECV ≤ 2 %) or simultaneously considering both cell lines (R2 = 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.
KW - FT-MIR spectroscopy
KW - GFP
KW - High-throughput analysis
KW - Reporter genes
UR - http://www.scopus.com/inward/record.url?scp=85027920067&partnerID=8YFLogxK
U2 - 10.1007/s00216-015-8983-9
DO - 10.1007/s00216-015-8983-9
M3 - Article
C2 - 26329279
AN - SCOPUS:85027920067
SN - 1618-2642
VL - 407
SP - 8097
EP - 8108
JO - Analytical and Bioanalytical Chemistry
JF - Analytical and Bioanalytical Chemistry
IS - 26
ER -