High-throughput microfluidic single-cell RT-qPCR

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 108; no. 34; pp. 13999 - 14004
• Main Authors: White, Adam K, VanInsberghe, Michael, Petriv, Oleh I, Hamidi, Mani, Sikorski, Darek, Marra, Marco A, Piret, James, Aparicio, Samuel, Hansen, Carl L
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 23.08.2011; National Acad Sciences
• Subjects: Biological Sciences; Breast cancer; breast neoplasms; Cell Line; Cell lines; Cells; Embryonic stem cells; Gene expression; Gene Expression Regulation; High-Throughput Screening Assays - methods; Humans; K562 cells; messenger RNA; Microfluidics - methods; MicroRNA; MicroRNAs - genetics; MicroRNAs - metabolism; Molecules; Physical Sciences; Polymorphism, Single Nucleotide - genetics; Reproducibility of Results; reverse transcriptase polymerase chain reaction; Reverse Transcriptase Polymerase Chain Reaction - methods; reverse transcription; Ribonucleic acid; RNA; RNA, Messenger - genetics; RNA, Messenger - metabolism; Single cell analysis; Single-Cell Analysis - methods; Stem cells; transcription (genetics)
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1019446108

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.