Advanced microRNA-based cancer diagnostics using amplified time-gated FRET

Xue Qiu (1, 2, 3) , Jingyue Xu (1, 2, 3) , Jiajia Guo (1, 2, 3) , Akram Yahia-Ammar (1, 3) , Nikiforos-Ioannis Kapetanakis (4, 2) , Isabelle Duroux-Richard (5) , Julia J. Unterluggauer (6) , Nicole Golob-Schwarzl (6) , Christophe Regeard (7, 1) , Catherine Uzan (8, 9, 2) , Sebastien Gouy (8, 10) , Michael Dubow (7, 1) , Johannes Haybaeck (6, 11, 12) , Florence Apparailly (5, 13) , Pierre Busson (4, 2) , Niko Hildebrandt (1, 3)

1 I2BC - Institut de Biologie Intégrative de la Cellule
2 Université Paris-Saclay
3 NANO - NanoBioPhotonics
4 UMR8126 - Signalisation, noyaux et innovations en cancérologie
5 Cellules Souches, Plasticité Cellulaire, Médecine Régénératrice et Immunothérapies (IRMB)
6 Medical University of Graz
7 LGBMB - Génomique et Biodiversité microbienne des biofilms
8 IGR - Institut Gustave Roussy
9 CRSA - Centre de Recherche Saint-Antoine
10 UMR 1030 - Radiothérapie moléculaire
11 OVGU - Otto-von-Guericke-Universität Magdeburg = Otto-von-Guericke University [Magdeburg]
12 IMU - Innsbruck Medical University = Medizinische Universität Innsbruck
13 CHU Montpellier

MicroRNAs (miRNAs) play an important role in cellular functions and in the development and progression of cancer. Precise quantification of endogenous miRNAs from different clinical patient and control samples combined with a one-to-one comparison to standard technologies is a challenging but necessary endeavor that is largely neglected by many emerging fluorescence technologies. Here, we present a simple, precise, sensitive, and specific ratiometric assay for absolute quantification of miRNAs. Isothermally amplified time-gated Forster resonance energy transfer (TG-FRET) between Tb donors and dye acceptors resulted in miRNA assays with single-nucleotide variant specificity and detection limits down to 4.2 +/- 0.5 attomoles. Quantification of miR-21 from human tissues and plasma samples revealed the relevance for breast and ovarian cancer diagnostics. Analysis of miR-132 and miR-146a from acute monocytic leukemia cells (THP-1) demonstrated the broad applicability to different miRNAs and other types of clinical samples. Direct comparison to the gold standard RT-qPCR showed advantages of amplified TG-FRET concerning precision and specificity when quantifying low concentrations of miRNAs as required for diagnostic applications. Our results demonstrate that a careful implementation of rolling circle amplification and TG-FRET into one straightforward nucleic acid detection method can significantly advance the possibilities of miRNA-based cancer diagnostics and research.

Mots clés: assay; isothermal amplification; rolling circle amplification; strategy

Domaines: Sciences du Vivant [q-bio]

Xue Qiu, Jingyue Xu, Jiajia Guo, Akram Yahia-Ammar, Nikiforos-Ioannis Kapetanakis, et al.. Advanced microRNA-based cancer diagnostics using amplified time-gated FRET. Chemical Science, 2018, 9 (42), pp.8046--8055. ⟨10.1039/c8sc03121e⟩. ⟨hal-02183316⟩