Quantitative evaluation of alternatively spliced mRNA isoforms by label-free real-time plasmonic sensing
Huertas, César S. (Institut Català de Nanociència i Nanotecnologia)
Carrascosa, Laura G. (Institut Català de Nanociència i Nanotecnologia)
Bonnal, Sophie (Universitat Pompeu Fabra)
Valcárcel, Juan (Universitat Pompeu Fabra)
Lechuga, Laura M (Institut Català de Nanociència i Nanotecnologia)

Date:	2016
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.
Grants:	Ministerio de Ciencia e Innovación TEC2012-3428 Agència de Gestió d'Ajuts Universitaris i de Recerca 2014/SGR-624 Ministerio de Economía y Competitividad SEV-2013-0295
Rights:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, i la comunicació pública de l'obra, sempre que no sigui amb finalitats comercials, i sempre que es reconegui l'autoria de l'obra original. No es permet la creació d'obres derivades.
Language:	Anglès
Document:	Article ; recerca ; Versió acceptada per publicar
Subject:	Alternative splicing ; Mrna isosoforms ; SPR ; Biosensor ; Biomarker ; Diagnosis
Published in:	Biosensors & bioelectronics, Vol. 78 (April 2016) , p. 118-125, ISSN 1873-4235

DOI: 10.1016/j.bios.2015.11.023

Postprint 32 p, 1.1 MB

The record appears in these collections:
Research literature > UAB research groups literature > Research Centres and Groups (research output) > Experimental sciences > Catalan Institute of Nanoscience and Nanotechnology (ICN2)
Articles > Research articles
Articles > Published articles