title: Quantitative evaluation of alternatively spliced mRNA isoforms by label-free real-time plasmonic sensing.
abstract: Alternative splicing of mRNA precursors enables cells to generate different protein outputs from the same gene depending on their developmental or homeostatic status. Its deregulation is strongly linked to disease onset and progression. Current methodologies for monitoring alternative splicing demand elaborate procedures and often present difficulties in discerning between closely related isoforms, e.g. due to cross-hybridization during their detection. Herein, we report a general methodology using a Surface Plasmon Resonance (SPR) biosensor for label-free monitoring of alternative splicing events in real-time, without any cDNA synthesis or PCR amplification requirements. We applied this methodology to RNA isolated from HeLa cells for the quantification of alternatively spliced isoforms of the Fas gene, involved in cancer progression through regulation of programmed cell death. We demonstrate that our methodology is isoform-specific, with virtually no cross-hybridization, achieving limits of detection (LODs) in the picoMolar (pM) range. Similar results were obtained for the detection of the BCL-X gene mRNA isoforms. The results were independently validated by RT-qPCR, with excellent concordance in the determination of isoform ratios. The simplicity and robustness of this biosensor technology can greatly facilitate the exploration of alternative splicing biomarkers in disease diagnosis and therapy.
authors.affiliation: Nanobiosensors and Bioanalytical Applications Group, Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC and The Barcelona Institute of Science and Technology, CIBER-BBN, Campus UAB, Bellaterra, 08193 Barcelona, Spain.
authors.forename: César S
authors.initials: CS
authors.lastname: Huertas
authors.affiliation: Nanobiosensors and Bioanalytical Applications Group, Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC and The Barcelona Institute of Science and Technology, CIBER-BBN, Campus UAB, Bellaterra, 08193 Barcelona, Spain.
authors.forename: L G
authors.initials: LG
authors.lastname: Carrascosa
authors.affiliation: Centre de Regulació Genòmica and The Barcelona Institute of Science and Technology, 08003 Barcelona, Spain; Universitat Pompeu Fabra, 08003 Barcelona, Spain.
authors.forename: S
authors.initials: S
authors.lastname: Bonnal
authors.affiliation: Centre de Regulació Genòmica and The Barcelona Institute of Science and Technology, 08003 Barcelona, Spain; Universitat Pompeu Fabra, 08003 Barcelona, Spain; Institució Catalana de Recerca i Estudis Avançats, Spain.
authors.forename: J
authors.initials: J
authors.lastname: Valcárcel
authors.affiliation: Nanobiosensors and Bioanalytical Applications Group, Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC and The Barcelona Institute of Science and Technology, CIBER-BBN, Campus UAB, Bellaterra, 08193 Barcelona, Spain. Electronic address: laura.lechuga@cin2.es.
authors.forename: L M
authors.initials: LM
authors.lastname: Lechuga
mesh_terms: D017398:Alternative Splicing / Q000235:genetics*; D015374:Biosensing Techniques / Q000379:methods*; D018076:DNA, Complementary / Q000737:chemistry; D006801:Humans; D016133:Polymerase Chain Reaction; D059368:RNA Isoforms / Q000235:genetics / Q000302:isolation & purification*; D020349:Surface Plasmon Resonance / Q000379:methods*
chemical_list: D018076:DNA, Complementary; D059368:RNA Isoforms
keywords: Alternative splicing; Biomarker; Biosensor; Diagnosis; SPR; mRNA isosoforms