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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Quantitative profiling of integrin αvβ3 on single cells with quantum dot labeling to reveal the phenotypic heterogeneity of glioblastoma
TL;DR: A novel protocol for the quantitative analysis of surface proteins from irregular single cells is provided, providing a clue for understanding the heterogeneity of tumor cells on the basis of molecular phenotypes and has potential significance in guiding targeted therapies for cancers.
Proceedings ArticleDOI
Component-Oriented High-level Synthesis for Continuous-Flow Microfluidics Considering Hybrid-Scheduling
TL;DR: This work proposes a component-oriented general device concept that enables precise description of operations and devices, and adapts well to technological updates, and proposes a layering algorithm together with a mathematical modeling method to synthesize binding and hybrid-scheduling solutions that support both fixed schedule and real-time decisions.
Patent
Sample indexing for single cells
Christina Chang,Christina Fan,Eleen Shum,Jody Martin,Nidhanjali Bansal,James Ghadiali,Katherine Lazaruk,Gretchen Yinbon Lam +7 more
TL;DR: In this paper, a sample indexing composition can comprise a protein binding reagent associated with a sample-indexing oligonucleotide, which can be used to identify the sample origin of cells.
Journal ArticleDOI
A focus on microfluidics and nanotechnology approaches for the ultra sensitive detection of microRNA.
TL;DR: N nanotechnology, microfluidics and liquid phase miRNA detection techniques that are highly sensitive and specific are described that can be used as point of care devices to rapidly detect low level miRNA in patient clinical samples to aid disease diagnosis and prognosis.
Journal ArticleDOI
Lab-on-a-chip mRNA purification and reverse transcription via a solid-phase gene extraction technique
TL;DR: This procedure was successfully used to extract, purify, and transcribe mRNA from rat glioblastoma cell spheroids in less than seven minutes and confirmed that the SPGE technique selectively captures and inherently purifies high-quality mRNA directly from biological material with no need for additional pre-processing steps.
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.
Journal ArticleDOI
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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