High-throughput microfluidic single-cell RT-qPCR
Adam K. White,Michael VanInsberghe,Oleh Petriv,Mani Hamidi,Darek Sikorski,Marco A. Marra,James M. Piret,Samuel Aparicio,Carl L. Hansen +8 more
TLDR
This work presents a fully integrated microfluidic device capable of performing high-precision RT-qPCR measurements of gene expression from hundreds of single cells per run, and shows that nanoliter volume processing reduced measurement noise, increased sensitivity, and provided single nucleotide specificity.Abstract:
A long-sought milestone in microfluidics research has been the development of integrated technology for scalable analysis of transcription in single cells Here we present a fully integrated microfluidic device capable of performing high-precision RT-qPCR measurements of gene expression from hundreds of single cells per run Our device executes all steps of single-cell processing, including cell capture, cell lysis, reverse transcription, and quantitative PCR In addition to higher throughput and reduced cost, we show that nanoliter volume processing reduced measurement noise, increased sensitivity, and provided single nucleotide specificity We apply this technology to 3,300 single-cell measurements of (i) miRNA expression in K562 cells, (ii) coregulation of a miRNA and one of its target transcripts during differentiation in embryonic stem cells, and (iii) single nucleotide variant detection in primary lobular breast cancer cells The core functionality established here provides the foundation from which a variety of on-chip single-cell transcription analyses will be developedread more
Citations
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Computational and experimental single cell biology techniques for the definition of cell type heterogeneity, interplay and intracellular dynamics.
TL;DR: A combination of experimental and computational advances has opened perspectives towards learning predictive multi-scale models of heterogeneous cell populations, yielding insights into topics such as immune cell differentiation and tumor cell type composition.
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Transcriptional mechanisms of cell fate decisions revealed by single cell expression profiling.
TL;DR: The need for single cell gene expression analysis is discussed to gain deeper insights into the transcriptional control of cell fate decisions, and the insights it has provided so far into transcriptional regulatory networks in development are considered.
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A Self-Digitization Dielectrophoretic (SD-DEP) Chip for High-Efficiency Single-Cell Capture, On-Demand Compartmentalization, and Downstream Nucleic Acid Analysis
Yuling Qin,Li Wu,Thomas Schneider,Gloria S. Yen,Jiasi Wang,Shihan Xu,Min Li,Amy L. Paguirigan,Jordan L. Smith,Jerald P. Radich,Robbyn K. Anand,Daniel T. Chiu +11 more
TL;DR: The design and fabrication of a self-digitization dielectrophoretic (SD-DEP) chip with simple components for single-cell manipulation and downstream nucleic acid analysis is presented and could be a promising and powerful tool in single- cell research for precise medicine.
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Rapid, quantitative, reverse transcription PCR in a polymer microfluidicchip
D. Curtis Saunders,Gregory L. Holst,Christopher R. Phaneuf,Nikita Pak,Matthew M. Marchese,Nicholas Sondej,Michael L. McKinnon,Craig R. Forest +7 more
TL;DR: This microfluidic qRT-PCR system offers a practical approach to rapid analysis (~1 h), combining the cost benefits of small reagent volumes with the simplicity of disposable polymer microchips and easy setup.
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In situ mRNA isolation from a microfluidic single-cell array using an external AFM nanoprobe
TL;DR: This integrated platform combines the non-destructive and precise-control of a single-cell mRNA probe with sealed microfluidic systems' capability of upstream sample processing and downstream multifunctional analysis to enable a versatile and powerful tool for biomedical research.
References
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MicroRNAs: Genomics, Biogenesis, Mechanism, and Function
TL;DR: Although they escaped notice until relatively recently, miRNAs comprise one of the more abundant classes of gene regulatory molecules in multicellular organisms and likely influence the output of many protein-coding genes.
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MicroRNA expression profiles classify human cancers
Jun Lu,Gad Getz,Eric A. Miska,Eric A. Miska,Ezequiel Alvarez-Saavedra,Justin Lamb,David Peck,Alejandro Sweet-Cordero,Alejandro Sweet-Cordero,Benjamin L. Ebert,Benjamin L. Ebert,Raymond H. Mak,Raymond H. Mak,Adolfo A. Ferrando,James R. Downing,Tyler Jacks,H. Robert Horvitz,H. Robert Horvitz,Todd R. Golub,Todd R. Golub,Todd R. Golub +20 more
TL;DR: A new, bead-based flow cytometric miRNA expression profiling method is used to present a systematic expression analysis of 217 mammalian miRNAs from 334 samples, including multiple human cancers, and finds the miRNA profiles are surprisingly informative, reflecting the developmental lineage and differentiation state of the tumours.
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Real-time quantification of microRNAs by stem–loop RT–PCR
Caifu Chen,Dana Ridzon,Adam Broomer,Zhaohui Zhou,Danny H. Lee,Julie T. Nguyen,Maura Barbisin,Nan Lan Xu,Vikram R. Mahuvakar,Mark R. Andersen,Kaiqin Lao,Kenneth J. Livak,Karl J. Guegler +12 more
TL;DR: A novel microRNA quantification method has been developed using stem–loop RT followed by TaqMan PCR analysis, which enables fast, accurate and sensitive miRNA expression profiling and can identify and monitor potential biomarkers specific to tissues or diseases.
Journal ArticleDOI
Monolithic microfabricated valves and pumps by multilayer soft lithography
TL;DR: An extension to the soft lithography paradigm, multilayersoft lithography, with which devices consisting of multiple layers may be fabricated from soft materials is described, to build active microfluidic systems containing on-off valves, switching valves, and pumps entirely out of elastomer.
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A mammalian microRNA expression atlas based on small RNA library sequencing.
Pablo Landgraf,Mirabela Rusu,Robert L. Sheridan,Alain Sewer,Alain Sewer,Nicola Iovino,Alexei A. Aravin,Sébastien Pfeffer,Amanda J. Rice,Alice O. Kamphorst,Markus Landthaler,Carolina Lin,Nicholas D. Socci,Leandro C. Hermida,Valerio Fulci,Sabina Chiaretti,Robin Foà,Julia Schliwka,Uta Fuchs,Astrid Novosel,Roman-Ulrich Müller,Roman-Ulrich Müller,Bernhard Schermer,Ute Bissels,Jason M. Inman,Quang Phan,Minchen Chien,David B. Weir,Ruchi Choksi,Gabriella De Vita,Daniela Frezzetti,Hans Ingo Trompeter,Veit Hornung,Grace Teng,Gunther Hartmann,Miklós Palkovits,Roberto Di Lauro,Peter Wernet,Giuseppe Macino,Charles E. Rogler,James W. Nagle,Jingyue Ju,F. Nina Papavasiliou,Thomas Benzing,Peter Lichter,Wayne Tam,Michael J. Brownstein,Andreas Bosio,Arndt Borkhardt,James J. Russo,Chris Sander,Mihaela Zavolan,Mihaela Zavolan,Thomas Tuschl +53 more
TL;DR: A relatively small set of miRNAs, many of which are ubiquitously expressed, account for most of the differences in miRNA profiles between cell lineages and tissues.
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