Quantitative evaluation of alternatively spliced mRNA isoforms by label-free real-time plasmonic sensing

Sanchez Huertas, C, Carrascosa, L, Bonnal, S, Valcarcel, J and Lechuga, L 2016, 'Quantitative evaluation of alternatively spliced mRNA isoforms by label-free real-time plasmonic sensing', Biosensors and Bioelectronics, vol. 78, pp. 118-125.


Document type: Journal Article
Collection: Journal Articles

Title Quantitative evaluation of alternatively spliced mRNA isoforms by label-free real-time plasmonic sensing
Author(s) Sanchez Huertas, C
Carrascosa, L
Bonnal, S
Valcarcel, J
Lechuga, L
Year 2016
Journal name Biosensors and Bioelectronics
Volume number 78
Start page 118
End page 125
Total pages 8
Publisher Elsevier BV
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.
Subject Medical Biotechnology Diagnostics (incl. Biosensors)
Keyword(s) Alternative splicing
Biomarker
Biosensor
Diagnosis
MRNA isosoforms
SPR
DOI - identifier 10.1016/j.bios.2015.11.023
Copyright notice © 2015 Elsevier B.V. All rights reserved.
ISSN 0956-5663
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