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

2015-10Journal article Restricted access
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dc.contributor.authorRosa, Filipa
dc.contributor.authorSales, Kevin C.
dc.contributor.authorCunha, Bernardo R.
dc.contributor.authorCouto, Andreia
dc.contributor.authorLopes, Marta B.
dc.contributor.authorCalado, Cecília
dc.date.accessioned2016-03-09T12:23:51Z
dc.date.available2016-03-09T12:23:51Z
dc.date.issued2015-10
dc.description.abstractReporter 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.pt_PT
dc.identifier.citationROSA, F. [et al.] - A comprehensive high-throughput FTIR spectroscopy-based method for evaluating the transfection event: estimating the transfection efficiency and extracting associated metabolic responses. Analytical and Bioanalytical Chemistry. ISSN.1618-2642. Vol. 407, N.º 26 (2015), pp. 8097-8109pt_PT
dc.identifier.doi10.1007/s00216-015-8983-9pt_PT
dc.identifier.issn1618-2642
dc.identifier.issn1618-2650
dc.identifier.urihttp://hdl.handle.net/10400.21/5811
dc.language.isoengpt_PT
dc.peerreviewedyespt_PT
dc.publisherSpringer Berlin Heidelbergpt_PT
dc.relation.publisherversionhttp://link.springer.com/article/10.1007/s00216-015-8983-9pt_PT
dc.subjectReporter genespt_PT
dc.subjectGFPpt_PT
dc.subjectFT-MIR spectroscopypt_PT
dc.subjectHigh-throughput analysispt_PT
dc.titleA comprehensive high-throughput FTIR spectroscopy-based method for evaluating the transfection event: estimating the transfection efficiency and extracting associated metabolic responsespt_PT
dc.typejournal article
dspace.entity.typePublication
oaire.citation.endPage8108pt_PT
oaire.citation.issue26pt_PT
oaire.citation.startPage8097pt_PT
oaire.citation.volume407pt_PT
person.familyNameCalado
person.givenNameCecília
person.identifier130332
person.identifier.ciencia-id9418-E320-3177
person.identifier.orcid0000-0002-5264-9755
person.identifier.ridE-2102-2014
person.identifier.scopus-author-id6603163260
rcaap.rightsclosedAccesspt_PT
rcaap.typearticlept_PT
relation.isAuthorOfPublicatione8577257-c64c-4481-9b2b-940fedb360cc
relation.isAuthorOfPublication.latestForDiscoverye8577257-c64c-4481-9b2b-940fedb360cc

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