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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Journal ArticleDOI
Highly multiplexed single-cell quantitative PCR.
TL;DR: Highly multiplexed microfluidic RT-qPCR fills a gap in current capabilities for single- cell analysis, providing a rapid and cost-effective approach for profiling panels of marker genes, thereby complementing single-cell genomics methods that are best suited for global analysis and discovery.
Journal ArticleDOI
Simple paired heavy- and light-chain antibody repertoire sequencing using endoplasmic reticulum microsomes
Praneeth Reddy Devulapally,Jörg Bürger,Jörg Bürger,Thorsten Mielke,Zoltán Konthur,Hans Lehrach,Marie-Laure Yaspo,Jörn Glökler,Hans-Jörg Warnatz +8 more
TL;DR: The method can be used for discovery of novel antigen-specific monoclonal antibodies by sequencing human CD19+ B cell IgM and IgG repertoires isolated from peripheral whole blood before and seven days after Td (Tetanus toxoid/Diphtheria toxoid) booster immunization.
Journal ArticleDOI
High-throughput microfluidic platform for adherent single cells non-viral gene delivery
Paola Occhetta,Chiara Malloggi,A. Gazaneo,Alberto Redaelli,Gabriele Candiani,Gabriele Candiani,Gabriele Candiani,Marco Rasponi +7 more
TL;DR: The devices were experimentally validated and allowed the trapping of individual human glioblastoma–astrocytoma epithelial-like cells (U87-MG) with a trapping efficacy of about 40%.
Journal ArticleDOI
Endovascular biopsy: Strategy for analyzing gene expression profiles of individual endothelial cells obtained from human vessels✩.
Zhengda Sun,Devon A. Lawson,Elizabeth Sinclair,Chih Yang Wang,Ming Derg Lai,Steven W. Hetts,Randall T. Higashida,Christopher F. Dowd,Van V. Halbach,Zena Werb,Hua Su,Daniel L Cooke +11 more
TL;DR: In this paper, the authors developed a strategy of achieving targeted collection of endothelial cells (ECs) by endovascular methods and analyzed the gene expression profiles of collected single ECs.
Journal ArticleDOI
Identification and validation of appropriate reference genes for qRT-PCR analysis in Corynebacterium glutamicum.
TL;DR: Using new reference genes from the commonly used housekeeping genes and from transcriptome data as candidate reference genes for normalization can improve the accuracy and reliability of the measurements of target gene expression levels obtained by qRT-PCR for C. glutamicum.
References
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Monolithic microfabricated valves and pumps by multilayer soft lithography
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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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