Toriello Nicholas M, Douglas Erik S, Thaitrong Numrin, Hsiao Sonny C, Francis Matthew B, Bertozzi Carolyn R, Mathies Richard A
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.
Proc Natl Acad Sci U S A. 2008 Dec 23;105(51):20173-8. doi: 10.1073/pnas.0806355106. Epub 2008 Dec 15.
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.
开发了一种用于分析单细胞基因表达的集成微器件。该系统捕获单个细胞，转录并扩增mRNA，并对感兴趣的产物进行定量分析。微器件的关键组件包括集成纳升计量泵、带有单细胞捕获垫的200纳升逆转录聚合酶链反应（RT-PCR）反应器，以及用于产物纯化和浓缩的亲和捕获基质，该基质与用于产物分析的微制造毛细管电泳分离通道相连。这些过程的高效微芯片集成能够在单细胞水平上对基因表达变异进行灵敏且定量的检测。该微器件用于测量单个Jurkat细胞中甘油醛-3-磷酸脱氢酶（GAPDH）基因的小干扰RNA（siRNA）敲低情况。单细胞测量表明存在两个不同的细胞群体，其沉默程度为中等（约50%）或完全（约0%）。这种单细胞内基因表达和沉默的随机变异被传统的大量测量所掩盖。
