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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Rapid and continuous magnetic separation in droplet microfluidic devices
TL;DR: A droplet microfluidic method to extract molecules of interest from a droplet in a rapid and continuous fashion by first marginalizing functionalized super-paramagnetic beads within the droplet using a magnetic field, and then splitting the droplets into one droplet containing the majority of magnetic beads and one droplets containing the minority fraction.
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Allele-specific detection of single mRNA molecules in situ.
TL;DR: A method for fluorescence in situ identification of individual mRNA molecules, allowing quantitative and accurate measurement, in single cells, of allele-specific transcripts that differ by only a few nucleotides, is described.
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Microfluidic isolation of nucleic acids.
TL;DR: The techniques presented include using silica-based surfaces, functionalized paramagnetic beads, oligonucleotide-modified polymer surfaces, pH-dependent charged surfaces, Al2O3 membranes, and liquid-phase isolation to improve NA isolation and move it toward achieving 100% efficiencies.
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Identification and genetic analysis of cancer cells with PCR-activated cell sorting
TL;DR: PCR-activated cell sorting (PACS) provides a general new technical capability that expands the application space of cell sorting by enabling sorting based on cellular information not amenable to existing approaches.
Journal ArticleDOI
Real-time analysis and selection of methylated DNA by fluorescence-activated single molecule sorting in a nanofluidic channel
Benjamin Cipriany,Patrick J. Murphy,James A. Hagarman,Aline Cerf,David R. Latulippe,S.L. Levy,Jaime J. Benítez,Christine P. Tan,Juraj Topolancik,Paul D. Soloway,Harold G. Craighead +10 more
TL;DR: A proof-of-principle demonstration for a new method using a nanofluidic device that combines real-time detection and automated sorting of individual molecules based on their epigenetic state, which provides a workflow for color-multiplexed detection, sorting, and recovery of single molecules toward subsequent DNA sequencing.
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.
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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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