A microfluidic processor for gene expression profiling of single human embryonic stem cells

Abstract

The gene expression of human embryonic stem cells (hESC) is a critical aspect for understanding the normal and pathological development of human cells and tissues. Current bulk gene expression assays rely on RNA extracted from cell and tissue samples with various degree of cellular heterogeneity. These 'cell population averaging' data are difficult to interpret, especially for the purpose of understanding the regulatory relationship of genes in the earliest phases of development and differentiation of individual cells. Here, we report a microfluidic approach that can extract total mRNA from individual single-cells and synthesize cDNA on the same device with high mRNA-to-cDNA efficiency. This feature makes large-scale single-cell gene expression profiling possible. Using this microfluidic device, we measured the absolute numbers of mRNA molecules of three genes (B2M, Nodal and Fzd4) in a single hESC. Our results indicate that gene expression data measured from cDNA of a cell population is not a good representation of the expression levels in individual single cells. Within the G0/G1 phase pluripotent hESC population, some individual cells did not express all of the 3 interrogated genes in detectable levels. Consequently, the relative expression levels, which are broadly used in gene expression studies, are very different between measurements from population cDNA and single-cell cDNA. The results underscore the importance of discrete single-cell analysis, and the advantages of a microfluidic approach in stem cell gene expression studies.

High-throughput microfluidic single-cell RT-qPCR

“…Considerable effort over the last decade has been directed toward developing integrated and scalable singlecell genetic analysis on chip (7,8). Thus, many of the basic functionalities for microfluidic single-cell gene expression analysis have been demonstrated in isolation, including cell manipulation and trapping (9, 10), RNA purification and cDNA synthesis (11)(12)(13), and microfluidic qPCR (14) following off-chip cell isolation, cDNA synthesis, and preamplification. In particular, microfluidic qPCR devices (Biomark Dynamic Array, Fluidigm) have recently been applied to single-cell studies (15,16).…”

Single-cell technologies in environmental omics

“…Physical phenomena such as diffusion and heat transfer take place quickly in small volumes, speeding up the whole process. A small droplet volume facilitates a high sample concentration in the droplet (Zhong et al, 2008), and more efficient PCR and MDA reactions (Marcy et al, 2007). Microdroplet technology makes it possible to reach high concentrations of cell-secreted molecules, enabling rapid detection of droplets that contain cells producing molecules of interest Brouzes et al, 2009).…”

High-throughput Protein Expression Generator Using a Microfluidic Platform

“…2. Expose the silicone molds to chlorotrimethylsilane (TMCS) vapor for 10 min to promote elastomer release after the baking steps 9 . 3.…”