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 (≈50%) or complete (≈0%) silencing. This stochastic variation in gene expression and silencing within single cells is masked by conventional bulk measurements.

[1]  E. Petricoin,et al.  Laser Capture Microdissection , 1996, Science.

[2]  Bo Huang,et al.  Counting Low-Copy Number Proteins in a Single Cell , 2007, Science.

[3]  Michael G. Roper,et al.  A fully integrated microfluidic genetic analysis system with sample-in–answer-out capability , 2006, Proceedings of the National Academy of Sciences.

[4]  T. Golos,et al.  Stable plasmid-based siRNA silencing of gene expression in human embryonic stem cells. , 2005, Stem cells and development.

[5]  R. Mathies,et al.  Radial capillary array electrophoresis microplate and scanner for high-performance nucleic acid analysis. , 1999, Analytical chemistry.

[6]  D. J. Harrison,et al.  Micromachining a Miniaturized Capillary Electrophoresis-Based Chemical Analysis System on a Chip , 1993, Science.

[7]  Robert H Singer,et al.  Materials and Methods Som Text Figs. S1 to S8 References and Notes Dynamics of Single Mrnps in Nuclei of Living Cells , 2022 .

[8]  Stellan Hjertén,et al.  High-performance electrophoresis : Elimination of electroendosmosis and solute adsorption , 1985 .

[9]  S. Lillard,et al.  A qualitative look at multiplex gene expression of single cells using capillary electrophoresis , 2005, Electrophoresis.

[10]  R. Mathies,et al.  Self-assembled cellular microarrays patterned using DNA barcodes. , 2007, Lab on a chip.

[11]  T. Burdon,et al.  Oct‐4 Knockdown Induces Similar Patterns of Endoderm and Trophoblast Differentiation Markers in Human and Mouse Embryonic Stem Cells , 2004, Stem cells.

[12]  P. Swain,et al.  Gene Regulation at the Single-Cell Level , 2005, Science.

[13]  R. Mathies,et al.  Integrated affinity capture, purification, and capillary electrophoresis microdevice for quantitative double-stranded DNA analysis. , 2007, Analytical chemistry.

[14]  Chaoyong James Yang,et al.  High-throughput single copy DNA amplification and cell analysis in engineered nanoliter droplets. , 2008, Analytical chemistry.

[15]  N. Friedman,et al.  Stochastic protein expression in individual cells at the single molecule level , 2006, Nature.

[16]  William H. Grover,et al.  Monolithic membrane valves and diaphragm pumps for practical large-scale integration into glass microfluidic devices , 2003 .

[17]  Richard A Mathies,et al.  Programmable cell adhesion encoded by DNA hybridization. , 2006, Angewandte Chemie.

[18]  C. Bertozzi,et al.  Cell surface engineering by a modified Staudinger reaction. , 2000, Science.

[19]  Richard A Mathies,et al.  Microfabricated bioprocessor for integrated nanoliter-scale Sanger DNA sequencing. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[20]  P. Andrews,et al.  Specific Knockdown of Oct4 and β2‐microglobulin Expression by RNA Interference in Human Embryonic Stem Cells and Embryonic Carcinoma Cells , 2004, Stem cells.

[21]  X. Xie,et al.  Probing Gene Expression in Live Cells, One Protein Molecule at a Time , 2006, Science.

[22]  Ji Huang,et al.  [Serial analysis of gene expression]. , 2002, Yi chuan = Hereditas.

[23]  R. Mathies,et al.  Monolithic integrated microfluidic DNA amplification and capillary electrophoresis analysis system , 2000 .

[24]  R. Mathies,et al.  Multichannel reverse transcription-polymerase chain reaction microdevice for rapid gene expression and biomarker analysis. , 2006, Analytical chemistry.

[25]  W. Lam,et al.  DNA-coated AFM cantilevers for the investigation of cell adhesion and the patterning of live cells. , 2008, Angewandte Chemie.

[26]  Stephen R. Quake,et al.  Microfluidic Digital PCR Enables Multigene Analysis of Individual Environmental Bacteria , 2006, Science.

[27]  Yi Liu,et al.  Single-Cell Gene Expression Profiling , 2022 .

[28]  Jared E. Toettcher,et al.  Stochastic Gene Expression in a Lentiviral Positive-Feedback Loop: HIV-1 Tat Fluctuations Drive Phenotypic Diversity , 2005, Cell.

[29]  S. Lillard,et al.  Measurement of single-cell gene expression using capillary electrophoresis. , 2001, Analytical chemistry.

[30]  Brian N. Johnson,et al.  An integrated nanoliter DNA analysis device. , 1998, Science.

[31]  Richard A Mathies,et al.  Inline injection microdevice for attomole-scale sanger DNA sequencing. , 2007, Analytical chemistry.

[32]  W. Gerald,et al.  Gene expression profiling in single cells within tissue , 2005, Nature Methods.

[33]  Jeffrey W. Smith,et al.  Stochastic Gene Expression in a Single Cell , 2022 .

[34]  Ronald W. Davis,et al.  Quantitative Monitoring of Gene Expression Patterns with a Complementary DNA Microarray , 1995, Science.