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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The Human Cell Atlas
Aviv Regev,Aviv Regev,Aviv Regev,Sarah A. Teichmann,Sarah A. Teichmann,Sarah A. Teichmann,Eric S. Lander,Eric S. Lander,Eric S. Lander,Ido Amit,Christophe Benoist,Ewan Birney,Bernd Bodenmiller,Bernd Bodenmiller,Peter J. Campbell,Peter J. Campbell,Piero Carninci,Menna R. Clatworthy,Hans Clevers,Bart Deplancke,Ian Dunham,James Eberwine,Roland Eils,Roland Eils,Wolfgang Enard,Andrew Farmer,Lars Fugger,Berthold Göttgens,Nir Hacohen,Nir Hacohen,Muzlifah Haniffa,Martin Hemberg,Seung K. Kim,Paul Klenerman,Paul Klenerman,Arnold R. Kriegstein,Ed S. Lein,Sten Linnarsson,Emma Lundberg,Emma Lundberg,Joakim Lundeberg,Partha P. Majumder,John C. Marioni,John C. Marioni,John C. Marioni,Miriam Merad,Musa M. Mhlanga,Martijn C. Nawijn,Mihai G. Netea,Garry P. Nolan,Dana Pe'er,Anthony Phillipakis,Chris P. Ponting,Stephen R. Quake,Wolf Reik,Wolf Reik,Wolf Reik,Orit Rozenblatt-Rosen,Joshua R. Sanes,Rahul Satija,Ton N. Schumacher,Alex K. Shalek,Alex K. Shalek,Alex K. Shalek,Ehud Shapiro,Padmanee Sharma,Jay W. Shin,Oliver Stegle,Michael R. Stratton,Michael J. T. Stubbington,Fabian J. Theis,Matthias Uhlen,Matthias Uhlen,Alexander van Oudenaarden,Allon Wagner,Fiona M. Watt,Jonathan S. Weissman,Barbara J. Wold,Ramnik J. Xavier,Nir Yosef,Nir Yosef,Human Cell Atlas Meeting Participants +81 more
TL;DR: An open comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease.
Journal ArticleDOI
Single-cell sequencing-based technologies will revolutionize whole-organism science
TL;DR: The unabated progress in next-generation sequencing technologies is fostering a wave of new genomics, epigenomics, transcriptomics and proteomics technologies, enabling high-throughput, multi-dimensional analyses of individual cells that will produce detailed knowledge of the cell lineage trees of higher organisms, including humans.
Journal ArticleDOI
Single-cell genome sequencing: current state of the science
TL;DR: An overview of the current state of the field of single-cell genome sequencing is provided, focusing on the technical challenges of making measurements that start from a single molecule of DNA, and how some of these recent methodological advancements have enabled the discovery of unexpected new biology.
Journal ArticleDOI
Functional roles of enhancer RNAs for oestrogen-dependent transcriptional activation
Wenbo Li,Dimple Notani,Qi Ma,Bogdan Tanasa,Bogdan Tanasa,Esperanza Nunez,Aaron Yun Chen,Daria Merkurjev,Jie Zhang,Kenneth A. Ohgi,Xiaoyuan Song,Soohwan Oh,Hong-Sook Kim,Christopher K. Glass,Michael G. Rosenfeld +14 more
TL;DR: It is reported that in human breast cancer cells 17β-oestradiol (E2)-bound oestrogen receptor α (ER-α) causes a global increase in eRNA transcription on enhancers adjacent to E2-upregulated coding genes, indicating that eRNAs are likely to have important functions in many regulated programs of gene transcription.
Journal ArticleDOI
Advances in microfluidic materials, functions, integration, and applications.
TL;DR: The successful demonstration of electrophoresis and electroosmotic pumping in a microfluidic device provided a nonmechanical method for both fluid control and separation, and integration of multiple processes can be highly enabling for many applications.
References
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Mutational evolution in a lobular breast tumour profiled at single nucleotide resolution
Sohrab P. Shah,Ryan D. Morin,Jaswinder Khattra,Leah M Prentice,Trevor J. Pugh,Angela Burleigh,Allen Delaney,Karen A. Gelmon,Ryan Guliany,Janine Senz,Christian Steidl,Robert A. Holt,Steven J.M. Jones,Mark G. F. Sun,Gillian Leung,Richard A. Moore,Tesa M. Severson,Greg Taylor,Andrew E. Teschendorff,Kane Tse,Gulisa Turashvili,Richard Varhol,René L. Warren,Peter H. Watson,Yongjun Zhao,Carlos Caldas,David G. Huntsman,Martin Hirst,Marco A. Marra,Samuel Aparicio +29 more
TL;DR: The data show that single nucleotide mutational heterogeneity can be a property of low or intermediate grade primary breast cancers and that significant evolution can occur with disease progression, and two new RNA-editing events that recode the amino acid sequence of SRP9 and COG3 are revealed.
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Characterization of human embryonic stem cell lines by the International Stem Cell Initiative
Oluseun Adewumi,Behrouz Aflatoonian,Lars Ährlund-Richter,Michal Amit,Peter W. Andrews,Gemma Beighton,Paul Bello,Nissim Benvenisty,Lorraine S. Berry,Simon Bevan,Barak Blum,Justin Brooking,Kevin G. Chen,Andre Bh Choo,Gary A. Churchill,Marie Corbel,Ivan Damjanov,John S Draper,Petr Dvorak,Petr Dvorak,Katarina Emanuelsson,Roland A. Fleck,Angela Ford,Karin Astrid Maria Gertow,Karin Astrid Maria Gertow,Marina Gertsenstein,Paul J. Gokhale,Rebecca S. Hamilton,Alex Hampl,Alex Hampl,Lyn Healy,Outi Hovatta,Johan Hyllner,Marta P. Imreh,Marta P. Imreh,Joseph Itskovitz-Eldor,Jamie P. Jackson,Jackie Johnson,Mark Jones,Kehkooi Kee,Benjamin L. King,Barbara B. Knowles,Majlinda Lako,Franck Lebrin,Barbara S. Mallon,Daisy Manning,Yoav Mayshar,Ronald D.G. McKay,Anna E. Michalska,Milla Mikkola,Masha Mileikovsky,Stephen L. Minger,Harry Moore,Christine L. Mummery,Andras Nagy,Norio Nakatsuji,Carmel M. O’Brien,Steve Oh,Cia Olsson,Timo Otonkoski,Kye-Yoon Park,Robert Passier,Hema Patel,Minal Patel,Roger A. Pedersen,Martin F. Pera,Marian S Piekarczyk,Renee A. Reijo Pera,Benjamin Reubinoff,Allan J. Robins,Janet Rossant,Peter J. Rugg-Gunn,Peter J. Rugg-Gunn,Thomas C Schulz,Henrik Semb,Eric S Sherrer,Henrike Siemen,Glyn Stacey,Miodrag Stojkovic,Hirofumi Suemori,Jin P. Szatkiewicz,Tikva Turetsky,Timo Tuuri,Steineke van den Brink,Kristina Vintersten,Sanna Vuoristo,Dorien Ward,Thomas A Weaver,Lesley Young,Weidong Zhang +89 more
TL;DR: The International Stem Cell Initiative characterized 59 human embryonic stem cell lines from 17 laboratories worldwide and found that despite diverse genotypes and different techniques used for derivation and maintenance, all lines exhibited similar expression patterns for several markers ofhuman embryonic stem cells.
Microfluidic Large Scale Integration
TL;DR: High-density microfluidic chips that contain plumbing networks with thousands of micromechanical valves and hundreds of individually addressable chambers are developed to construct the microfluidity analog of a comparator array and a microfluidsic memory storage device whose behavior resembles random-access memory.
Journal ArticleDOI
Resolution of Cell Fate Decisions Revealed by Single-Cell Gene Expression Analysis from Zygote to Blastocyst
Guoji Guo,Mikael Huss,Guo Qing Tong,Chaoyang Wang,Li Li Sun,Neil D. Clarke,Paul Robson,Paul Robson +7 more
TL;DR: It is shown that in the morula, blastomeres coexpress transcription factors specific to different lineages, but by the 64-cell stage three cell types can be clearly distinguished according to their quantitative expression profiles.
Journal ArticleDOI
Feeder-independent culture of human embryonic stem cells.
Tenneille Ludwig,Tenneille Ludwig,Veit Bergendahl,Mark E. Levenstein,Junying Yu,Mitchell D. Probasco,James A. Thomson +6 more
TL;DR: Modifications to the medium (mTeSR1) that include the use of animal-sourced proteins (bovine serum albumin (BSA) and Matrigel) and cloned zebrafish basic fibroblast growth factor (zbFGF) are described.
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Evan Z. Macosko,Evan Z. Macosko,Anindita Basu,Anindita Basu,Rahul Satija,Rahul Satija,James Nemesh,James Nemesh,Karthik Shekhar,Melissa Goldman,Melissa Goldman,Itay Tirosh,Allison R. Bialas,Nolan Kamitaki,Nolan Kamitaki,Emily M. Martersteck,John J. Trombetta,David A. Weitz,Joshua R. Sanes,Alex K. Shalek,Alex K. Shalek,Alex K. Shalek,Aviv Regev,Aviv Regev,Aviv Regev,Steven A. McCarroll,Steven A. McCarroll +26 more