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. |
Note: |
Número d'acord de subvenció MICINN/TEC2012-3428 |
Note: |
Número d'acord de subvenció AGAUR/2014/SGR-624 |
Note: |
Número d'acord de subvenció MINECO/SEV-2013-0295 |
Rights: |
Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial 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 ; acceptedVersion |
Subject: |
Alternative splicing ;
Mrna isosoforms ;
SPR ;
Biosensor ;
Biomarker ;
Diagnosis |
Published in: |
Biosensors and bioelectronics, Vol. 78 (April 2016) , p. 118-125, ISSN 1873-4235 |
DOI: 10.1016/j.bios.2015.11.023
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Record created 2019-09-23, last modified 2021-01-25