title: High-throughput microfluidic single-cell RT-qPCR.
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 developed.
authors.affiliation: Centre for High-Throughput Biology, University of British Columbia, Vancouver, BC, Canada V6T 1Z4.
authors.forename: Adam K
authors.initials: AK
authors.lastname: White
authors.forename: Michael
authors.initials: M
authors.lastname: VanInsberghe
authors.forename: Oleh I
authors.initials: OI
authors.lastname: Petriv
authors.forename: Mani
authors.initials: M
authors.lastname: Hamidi
authors.forename: Darek
authors.initials: D
authors.lastname: Sikorski
authors.forename: Marco A
authors.initials: MA
authors.lastname: Marra
authors.forename: James
authors.initials: J
authors.lastname: Piret
authors.forename: Samuel
authors.initials: S
authors.lastname: Aparicio
authors.forename: Carl L
authors.initials: CL
authors.lastname: Hansen
mesh_terms: D002460:Cell Line; D005786:Gene Expression Regulation; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism; D044085:Microfluidics / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D035683:MicroRNAs; D012333:RNA, Messenger