BEAD-BASED MICROFLUIDIC RT-QPCR ANALYSIS OF SINGLE CANCER CELLS H. Sun1, 2, T. Olsen1, J. Zhu1, B. Ponnaiya3, S. Amundson4, D. Brenner3, 4, J. Tao2, and Q. Lin1* 1Department of Mechanical Engineering, 3Center for Radiological Research, and 4Department of Radiation Oncology, Columbia University, New York, NY, USA 2Department of Mechatronics Engineering, Harbin Institute of Technology, Harbin, Heilongjiang, China ABSTRACT Single- microfluidic RT-qPCR can enable assays of small volumes, with efficient thermal cycling, and reduced costs. Such devices currently use solution-based methods, which, while well established, do not allow efficient manipulation of samples and reagents or retrieval of reaction products for real-time single-cell genetic analysis. Employing a novel microbead-based approach and for the first time, we present a microchip that integrates all steps for genetic analysis of an individual cell. The effects of the chemotherapy drug (MMS) on the induction of CDKN1A in single human cancer cells (MCF-7) were assayed, demonstrating the potential of the device for single-cell analysis. KEYWORDS: Single-cell, bead-based, microfluidics, RT-qPCR

INTRODUCTION Gene expression profiling at single-cell level is critical to understanding substantial variations among cells in heterogeneous populations [1]. Quantitative reverse transcription polymerase chain reaction (RT- qPCR) allows effective single-cell gene expression analysis with high specificity and sensitivity, and large dynamic range. Conventional RT-qPCR platforms are labor-intensive and high in reagent consumption, and do not have adequate accuracies for low-abundance transcripts [2]. Single-cell microfluidic RT-qPCR can enable assays of small volumes, with efficient thermal cycling, and reduced costs [3]. Such devices currently use solution-based methods, which, while well established, do not allow efficient manipulation of samples and reagents or retrieval of reaction products for real-time analysis of singe-cell gene expression profiling. We address these issues using a microbead-based approach and present a microfluidic device using this approach to integrate all steps for single-cell transcriptional profiling, including immobilization and lysis of the cell, as well as purification, reverse transcription (RT) and quantitative real-time PCR (qPCR) of messenger RNA (mRNA). As such, all reactions in the multi- step assay to be completed in a single chamber, thereby simplifying the device design, fabrication and operation, as well as eliminating the potential loss or contamination of the analyte.

PRINCIPLE AND DEVICE The device is capable of cell-trapping, cell lysis and bead-based RT-qPCR. The mRNA is captured on microbeads via base pairing between the polyA tails of the mRNA and the oligo(dT)25 residues covalently coupled to bead surfaces (Fig. 1A). Next, through reverse transcription (RT), the complementary DNA (cDNA) to the mRNA is generated. This is followed by quantitative real-time PCR (qPCR), in which teh cDNA is amplified and detected using a hydrolysis probe/primer set. The device consists of a temperature control chip with an integrated heater and temperature sensor, and a polydimethylsiloxane (PDMS) microchamber with a neck-shaped constriction located between two pneumatically controlled microvalves for trapping a single cell (Fig. 1B). To minimize reagent evaporation, a polyethylene film is embedded in the PDMS above the microchamber to serve as a barrier. During experiments (Fig. 1C), cells are introduced to the device, with a single cell trapped in the constriction under control of the microvalves, and lysed. The cell mRNA, now in lysate, is captured and undergoes RT on oligo (dT)25-functionalized magnetic microbeads. The resulting complementary DNA (cDNA) is next amplified in the microchamber via qPCR. In qPCR, which occurs on the microbeads, the cleavage of the detecting probe (TaqMan) caused by new strand elongation separates the reporter and the quencher dyes, resulting in increased fluorescence, which is quantified at the end of each PCR cycle. The

978-0-9798064-7-6/µTAS 2014/$20©14CBMS-0001 811 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences October 26-30, 2014, San Antonio, Texas, USA single-cell transcriptional profiling chip was fabricated using standard multi-layer soft lithography microfabrication techniques. A closed-loop RT-qPCR system (Fig. 2) including a digital multimeter, a DC power supply, a pressure regulator and an inverted epifluorescence microscope with a CCD camera, was employed to perform typical RT (10 min at 25 °C and 50 min at 42 °C) and PCR (15 s at 95 °C and 1 min at 60 °C). The fluorescent intensity of the reaction was measured from images acquired at the end of each PCR cycle.

Figure 1: The microfluidic RT-qPCR device: (A) Bead-based RT-qPCR principle. Oligo (dT)25 bead (2.80.2 m) is composed by a superparamagnetic particle and a polymer shell. (B) Schematic of the device. (C) Operation of the device.

Figure 2: Schematic of the experimental set-up for Figure 3: Time-resolved tracking of the closed-loop RT-qPCR with a photograph of a chamber temperature. A typical RT and a packaged device .in the inset. 35-cycle PCR processes are fully integrated in the platform. RESULTS AND DISCUSSION We first evaluated the accuracy and precision of the system over the course of RT and PCR cycles (Fig. 3). The results indicate that the chamber temperature can be controlled to produce accurate and rapid amplification reactions. Then, we calibrated the microscope imaging system by testing the response of the camera using a series of fluorophore dilutions (Fig. 4A). Next, we validated on-chip RT-qPCR using XenoRNA (a synthetic RNA transcript) templates (Fig. 4B). The mean fluorescent intensity value of three XenoRNA samples after 35-cycles of PCR was 2.7±0.2 compared to 0±0.05 with the no-template control (NTC). This indicates that there was a significant amplification of XenoRNA templates and negligible amplification of the NTC. In addition, we investigated the bead quantity effect on RT-qPCR (Fig. 4C), the mRNA capture efficiency (Fig. 4D) and on-chip PCR efficiency (Fig. 4E). As the quantity of beads in the chamber increased from 20 to 197 g (nominal mass, based on mean diameter (2.8 µm) and manufacturer-supplied density (1.6 g/cm3) for the beads in regular packing), ΔRn, the magnitude of fluorescence signal generated from PCR, initially increased and then decreased after reaching a maximum 812 at an optimum bead quantity of 3.8 µg (Fig. 4C). From Fig. 4D, it can be seen that all the XenoRNA were captured by 37.5×105 beads and free RNA templates were not detected in the waste. From Fig. 4E, the PCR efficiency was found to be 99.7% for on-chip PCR and 80.2% for in-tube bead-based PCR and 83.9% for in-tube solution phase PCR. Based on these results, we performed fully integrated single-cell transcriptional profiling.

Figure 4: (A) Microscope characterization. (B) On -chip RT-PCR validation. (C) Bead quantity optimization. (D) RNA capture efficiency test. (E) Efficiency testing of in-tube and on-chip PCR. (F) Fully integrated on-chip single-cell RT-qPCR.

The expression levels of cyclin-dependent kinase inhibitor 1A (CDKN1A) gene of single MCF-7 cell were measured (Fig. 4F). For untreated (red line) and MMS-treated single cells (blue line), the mean values of threshold cycle number (Cq) were 32.26 and 26.78, respectively, indicating that MMS upregulated the expression level of CDKN1A significantly. These results demonstrate the potential of the device for efficient, integrated RT-qPCR analysis of single cells.

CONCLUSION In this work, we have developed a bead-based microfluidic device for integrated RT-qPCR at the single cell level. We first tested the mRNA capture efficiency by microbeads and evaluated the efficiency, sensitivity and repeatability of the microdevice. Then, the on-chip cell trapping and lysis efficiency were studied. On this basis, gene expression levels of MMS treated and untreated single MCF-7 cells were profiled in the device. The results demonstrated that our microfluidic approach can potentially be used for single-cell analysis in basic biological research and clinical diagnostics. ACKNOWLEDGEMENTS We gratefully acknowledge financial support from the National Institutes of Health (Award Nos. 5U19 AI067773 and 8R21GM104204). REFERENCE [1] Shalek A. K., et al. “Single-cell transcriptomics reveals bimodality in expression and splicing in immune cells,” Nature, 2013. [2] White A. K., et al. “High-throughput microfluidic single-cell RT-qPCR,” PNAS, 2011, 108(34): 13999-14004. [3] Verpoorte E., et al. “Microfluidic chips for clinical and forensic analysis,” Electrophoresis, 2002, 23(5): 677-712. CONTACT * Qiao Lin; phone: +1-212-854-3221; [email protected]

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