TY - JOUR
T1 - Advanced microRNA-based cancer diagnostics using amplified time-gated FRET
AU - Qiu, Xue
AU - Xu, Jingyue
AU - Guo, Jiajia
AU - Yahia-Ammar, Akram
AU - Kapetanakis, Nikiforos Ioannis
AU - Duroux-Richard, Isabelle
AU - Unterluggauer, Julia J.
AU - Golob-Schwarzl, Nicole
AU - Regeard, Christophe
AU - Uzan, Catherine
AU - Gouy, Sébastien
AU - Dubow, Michael
AU - Haybaeck, Johannes
AU - Apparailly, Florence
AU - Busson, Pierre
AU - Hildebrandt, Niko
N1 - Publisher Copyright:
© 2018 The Royal Society of Chemistry.
PY - 2018/1/1
Y1 - 2018/1/1
N2 - 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 Förster 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.
AB - 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 Förster 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.
UR - http://www.scopus.com/inward/record.url?scp=85056109117&partnerID=8YFLogxK
U2 - 10.1039/c8sc03121e
DO - 10.1039/c8sc03121e
M3 - Article
AN - SCOPUS:85056109117
SN - 2041-6520
VL - 9
SP - 8046
EP - 8055
JO - Chemical Science
JF - Chemical Science
IS - 42
ER -