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Issue 17, 2015
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Stability analysis of chemically modified mRNA using micropattern-based single-cell arrays

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Abstract

The measurement of mRNA turnover in living cells plays an important role in the search for stable mRNA constructs for RNA-based therapies. Here we show that automated time-lapse microscopy combined with micropatterned arrays allows for efficient high-throughput monitoring of fluorescent reporter protein expression at the single-cell level. The fluorescence time courses after mRNA transfection yield the distribution of individual mRNA expression and degradation rates within a population. We compare mRNA constructs with combinations of 5′ and 3′ UTR sequences and find a systematic broadening and shift towards longer functional half-lives for UTR stabilized mRNA. At the same time the life time distribution of the destabilized EGFP reporter protein was found to be constant and narrowly distributed. Using mathematical modeling, we show that mRNA functional life-time predicts the time-integrated protein level, i.e. the area under the curve (AUC) of mRNA translation. Our approach paves the way for quantitative assessment of hitherto unexplored mRNA functional life time heterogeneity, possibly predicated on multiple mRNA secondary structures and its dependence on UTR sequences.

Graphical abstract: Stability analysis of chemically modified mRNA using micropattern-based single-cell arrays

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Publication details

The article was received on 30 Jun 2015, accepted on 15 Jul 2015 and first published on 16 Jul 2015


Article type: Paper
DOI: 10.1039/C5LC00749F
Citation: Lab Chip, 2015,15, 3561-3571
  • Open access: Creative Commons BY-NC license
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    Stability analysis of chemically modified mRNA using micropattern-based single-cell arrays

    M. Ferizi, C. Leonhardt, C. Meggle, M. K. Aneja, C. Rudolph, C. Plank and J. O. Rädler, Lab Chip, 2015, 15, 3561
    DOI: 10.1039/C5LC00749F

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