Integrated microfluidic bioprocessor for single-cell gene expression analysis.
An integrated microdevice is developed for the analysis of gene expression in single cells. The system captures a single cell, transcribes and amplifies the mRNA, and quantitatively analyzes the products of interest. The key components of the microdevice include integrated nanoliter metering pumps, a 200-nL RT-PCR reactor with a single-cell capture pad, and an affinity capture matrix for the purification and concentration of products that is coupled to a microfabricated capillary electrophoresis separation channel for product analysis. Efficient microchip integration of these processes enables the sensitive and quantitative examination of gene expression variation at the single-cell level. This microdevice is used to measure siRNA knockdown of the GAPDH gene in individual Jurkat cells. Single-cell measurements suggests the presence of 2 distinct populations of cells with moderate (approximately 50%) or complete (approximately 0%) silencing. This stochastic variation in gene expression and silencing within single cells is masked by conventional bulk measurements.
Toriello
Nicholas M
NM
University of California San Francisco/University of California Berkeley Joint Graduate Group in Bioengineering, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720, USA.
Douglas
Erik S
ES
Thaitrong
Numrin
N
Hsiao
Sonny C
SC
Francis
Matthew B
MB
Bertozzi
Carolyn R
CR
Mathies
Richard A
RA
D002477:Cells / Q000737:chemistry*; D046209:Electrophoresis, Microchip; D004867:Equipment Design; D020869:Gene Expression Profiling / Q000295:instrumentation* / Q000379:methods*; D006801:Humans; D019169:Jurkat Cells; D056656:Lab-On-A-Chip Devices; D008832:Microchemistry; D036103:Nanotechnology / Q000295:instrumentation*; D020133:Reverse Transcriptase Polymerase Chain Reaction