A Nanoliter-Scale Nucleic Acid Processor with Parallel Architecture Jong Wook Hong, Vincent Studer, Giao Hang, W French Andreson, Stephen R Quake presented by: Anna Shcherbina Michael Meyer

Goal: Use Single Cell To Establish cDNA Library Gene Expression Profile Motivation for Single-Cell mRNA/ DNA Extraction Primary cells hard to obtain in large quantities. Isolating cells from animals or patients results in a mixture of cell types. Epigenetic variation between cells with identical genotypes influences development.

Existing Technologies and their Limitations Affinity capture and elution of purified DNA from silicon microstructures Deep Reactive Ion Etching (DRIE) On the order of uL, not nL No parallelization No integration Microarrays measure expression of a few genes from a single cell Amplification process introduces distortion Require choice of finite set of possible transcripts Currently, cDNA library construction methods requires ,000 input cells.

Innovation: Microfluidic Chip to Sequentially Process nL Volumes and Isolate Cells Small-Volume Scaling Process Integration Fabricated by multi-layer soft lithography Lysing and purification performed directly on the chip. No pre/post-treatment needed. Compatible with many biological assays Protein crystallization nL -volume PCR FACS single-cell enzyme screening

Fig 1. a. Layout of microfluidic chip, version 1. Channels are 100 um wide. Fluidic ports are named; actuation ports are numbered Lysing buffer chamber cell chamber bead chamber b. Photograph of the in situ affinity column construction. Scale bar 200 um. c. Cell loaded into cell chamber before lysis step. Scale bar 100 um. (Hong et al.) mRNA Purification Chip Design affinity column

Performance & Sensitivity Assay Fig 2. RT-PCR analysis of isolated mRNA. RT-PCR products analyzed on 2% agarose gel loaded with 5% of reaction. (Hong et al.) Primers used to identify high abundance B-actin transcript and moderate abundance OZF transcript. 18 experiments performed. 5 of these used a single cell.

Chip Sensitivity Assay Results B-actin purified from cells in 14 out of 18 experiments Between 2-10 cells required to detect OZF mRNA signal Monotonically increasing relationship between band intensity and cell number in B- actin mRNA. No functional relationship observed in non-normalized data. Fig 3. RT-PCR products for both transcripts were analyzed on a 2% agarose gel, whose bands were quantified and normalized. Zero values indicate absence of detectable band in gel. (Hong et al.)

Parallelization Align several linear processors and use same cross-junction structures to load them simultaneously. Loading & Processing Flow Loading--fluid flows north/south Processing--fluid flows east/west, along each batch processor Customization DNA Purification Chip Architecture (advances) Fig 4. Temporal action of DNA isolation circuitry (Hong et al.)

Each processor hold 5 nL Volume of cells used: 1.6, 1.0, 0.4 nL remaining volume is reaction buffer and lysis buffer Full Chip and Experimental Setup - Characterization of Sensitivity Fig 5. Food-coloring reveals the interconnectivity of chip. (Hong et al.)

Fig 6. Verification of the successful recovery of E. coli genomic DNA. Samples have been PCR amplified. (Hong et al.) a. Undiluted E. coli culture Lane 1: 1.6 nL culture (~1120 cells) Lane 2: 1.0 nL culture (~700 cells) Lane 3: 0.4 nL culture (~280 cells) Lanes 4-6: Negative control (pure H 2 O) b. 1:10 dilutions Lanes 1,4,7: diluted 1.6 nL Lanes 2,5,8: diluted 1.0 nL Lanes 3,6,9: diltued 0.4 nL c. Intensity of gel bands DNA Yield Experimental Results

Potential Uses and Impact Increasing throughput in single-cell analysis Automation of reagent preparation for large cell populations industrial-scale microarray analysis Preparation step for environmental analysis or medical diagnostics Tool for microculture and analysis of slow-growing or unculturable bacteria Generation of subtractive libraries from pairs of single cells eliminate commonly expressed transcripts & enrich differentially expressed transcripts