Microfluidic single-cell mRNA isolation and analysis

被引:256
作者
Marcus, Joshua S.
Anderson, W. French
Quake, Stephen R.
机构
[1] CALTECH, Pasadena, CA 91125 USA
[2] Univ So Calif, Keck Sch Med, Gene Therapy Labs, Los Angeles, CA 90033 USA
关键词
D O I
10.1021/ac0519460
中图分类号
O65 [分析化学];
学科分类号
070302 ; 081704 ;
摘要
Single-cell gene expression analysis holds great promise for studying diverse biological systems, but methodology to process these precious samples in a reproducible, quantitative, and parallel fashion remains challenging. Here, we utilize microfluidics to isolate picogram and subpicogram mRNA templates, as well as to synthesize cDNA from these templates. We demonstrate single-cell mRNA isolation and cDNA synthesis, provide quantitative calibrations for each step in the process, and measure gene expression in individual cells. The techniques presented here form the foundation for highly parallel single-cell gene expression studies.
引用
收藏
页码:3084 / 3089
页数:6
相关论文
共 27 条
[1]  
Alberts B., 1994, MOL BIOL CELL
[2]   Gene expression profiling in single cells from the pancreatic islets of Langerhans reveals lognormal distribution of mRNA levels [J].
Bengtsson, M ;
Ståhlberg, A ;
Rorsman, P ;
Kubista, M .
GENOME RESEARCH, 2005, 15 (10) :1388-1392
[3]   3 ABUNDANCE CLASSES IN HELA-CELL MESSENGER-RNA [J].
BISHOP, JO ;
MORTON, JG ;
ROSBASH, M ;
RICHARDSON, M .
NATURE, 1974, 250 (5463) :199-204
[4]   Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays [J].
Bustin, SA .
JOURNAL OF MOLECULAR ENDOCRINOLOGY, 2000, 25 (02) :169-193
[5]   Single-cell transcript analysis of pancreas development [J].
Chiang, MK ;
Melton, DA .
DEVELOPMENTAL CELL, 2003, 4 (03) :383-393
[6]   A Microfabricated Rotary Pump [J].
Chou, Hou-Pu ;
Unger, Marc A. ;
Quake, Stephen R. .
BIOMEDICAL MICRODEVICES, 2001, 3 (04) :323-330
[7]   Widely expressed transcripts for chemokine receptor CXCR1 in identified glutamatergic, γ-aminobutyric acidergic, and cholinergic neurons and astrocytes of the rat brain:: A single-cell reverse transcription-multiplex polymerase chain reaction study [J].
Danik, M ;
Puma, C ;
Quirion, R ;
Williams, S .
JOURNAL OF NEUROSCIENCE RESEARCH, 2003, 74 (02) :286-295
[8]   A NOVEL FAMILY OF GENES ENCODING PUTATIVE PHEROMONE RECEPTORS IN MAMMALS [J].
DULAC, C ;
AXEL, R .
CELL, 1995, 83 (02) :195-206
[9]   A microfabricated fluorescence-activated cell sorter [J].
Fu, AY ;
Spence, C ;
Scherer, A ;
Arnold, FH ;
Quake, SR .
NATURE BIOTECHNOLOGY, 1999, 17 (11) :1109-1111
[10]   Systematic investigation of protein phase behavior with a microfluidic formulator [J].
Hansen, CL ;
Sommer, MOA ;
Quake, SR .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2004, 101 (40) :14431-14436