single-cell level gene expression profiling using microfluidic linear ...

SINGLE-CELL LEVEL GENE EXPRESSION PROFILING USING MICROFLUIDIC LINEAR AMPLIFICATION J.G. Kralj1, A. Player2, M.S. Munson1, S.P. Forry1, D. Petersen2, D. Edelman2, E. Kawasaki2, P. Meltzer2, L.E. Locascio1 1

National Institute of Standards and Technology (NIST), USA and National Cancer Insitute/Naitonal Institutes of Health (NCI/NIH), USA

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ABSTRACT We show single-cell level linear amplification of gene expression libraries immobilized on a microfluidic packed bed. This is a critical step in the Eberwine process, [1] which is used to amplify messenger RNA (mRNA) for gene expression profiling. Microarray assays comparing microfluidic and benchtop reactions showed equivalent levels of gene detection from approximately 5000 to 9000 transcripts (51 % to 97 % global gene expression), and concordance between methods above 90 %. KEYWORDS: Gene expression profiling, linear amplification, RNA, single cell INTRODUCTION Measuring gene expression from small samples is challenging because RNA is easily degraded and exists in small amounts (≈10 pg per cell, 1 % to 5 % as mRNA), capture and enzymatic reactions are performed under suboptimal conditions giving relatively poor yield, and transfer and purification steps result in sample loss. Yet small sample processing is critical to identify the role of microenvironments within tissues, thereby elucidating effects of individual/rare cells on whole tissue. Currently, there are two main approaches to small-sample gene expression analysis. Polymerase chain reaction (PCR)-based methods can provide rapid amplification down to single copies of genes, though the expression profile containing several thousand genes can be biased towards short-length transcripts, with misrepresentation of the transcriptome. This flaw makes PCR-based methods unsuitable for whole transcriptome studies. The Eberwine method [1] provides reliable representation of the transcriptome, though the method requires starting amounts of RNA of at least 10 ng (≈1000 cell equivalents). This concentration problem can be overcome by miniaturization, as demonstrated by on-chip mRNA capture/purification [2, 3] and reverse transcription (RT) of single cell mRNA to complementary DNA (cDNA) [4-6]. We also previously demonstrated linear amplification (in vitro transcription, IVT, Figure 1) of immobilized Jurkat T-cell cDNA libraries [7].

Figure 1. Amplification Scheme. Amplification of mRNA is achieved by converting to cDNA, then generating about 1000 antisense RNA (aRNA) copies using T7 RNA polymerase.

Twelfth International Conference on Miniaturized Systems for Chemistry and Life Sciences October 12 - 16, 2008, San Diego, California, USA 978-0-9798064-1-4/µTAS2008/$20©2008CBMS

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Here, we report IVT from single-cell cDNA amounts. We performed the IVT reaction using RNA from 2 to 1000 cell equivalents (20 pg to 10 ng total RNA). Once on device, the cDNA can be reused, whereas typical solution-phase amplifications are single-use. The packed-bed architecture also enables the integration of other chip-based mRNA handling techniques that reduce sample transfer losses and incorporate chip-based capture and RT steps. Each round of amplification gives ≈1000 copies of each cDNA template, which were reamplified on the benchtop to microgram quantities, and analyzed using electrophoresis, microarrays, and PCR. EXPERIMENTAL The device consisted of a multilayer poly(dimethylsiloxane) device with a sieve valve architecture [6] that allowed microsphere capture in the device (Figure 2).

Figure 2. Device Images. (A) The 9-channel device with fluidic and valve connections. (B) The columns are made by flowing a bead slurry through a sieve valve. The cDNA from 10 ng, 100 ng, and 1 μg Jurkat total RNA was benchtop synthesized onto T7-oligo(dT) functionalized microspheres using a reagent kit based on the Eberwine process [1]. Aliquots of 0.5 % to 2 % of each sample were injected and the beads captured. IVT reaction mixture was then pulsed over the column (10 nL/min average, 10 s at 100 nL/min, 90 s stop flow). All samples were reamplified on the benchtop, incorporating biotinylated bases and to bring the aRNA to μg quantities. Electrophoresis was used to characterize average template length. Microarrays were used to analyze 0.85 μg of biotin-labeled (for staining) aRNA. PCR was used to confirm the aRNA was genomic by testing for presence/absence of Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Isopropyl β-D-1-thiogalactopyranoside (ITPG) housekeeping genes. RESULTS AND DISCUSSION Product aRNA from device amplification had equivalent length distributions to benchtop amplification techniques (Figure 3). This suggests high processivity of the enzymes and optimal reaction conditions. Microarray expression studies comparing benchtop and microfluidic amplification methods showed good agreement, indicating no significant expression bias was introduced by the microfluidic amplification (Figure 3). Starting sample sizes from 1 Twelfth International Conference on Miniaturized Systems for Chemistry and Life Sciences October 12 - 16, 2008, San Diego, California, USA

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ng to 10 ng (100 to 1000 cell eq.) yielded 7348 to 9008 genes detected, or 76 % to 93 % of the global gene expression (GGE). Samples from 20 pg to 100 pg (2 to 10 cell eq.) yielded 5352 to 8513 genes detected, or 55 % to 88 % of the GGE. The number of concordant genes between benchtop and device exceeded 90 % for all cases. PCR amplification of GAPDH and IPTG genes was used to confirm the presence of genomic material for both amplification methods (data not shown).

Figure 3. Sample characterization. (A) Electropherograms of all samples show equivalent length distributions. (B) Microarray data comparing amplification methods (gene concordance at 95 % confidence in parantheses) with similar sample sizes show excellent agreement at all concentration. CONCLUSIONS Microfluidic IVT enables amplification of of single-cell cDNA samples. Integrating single-cell IVT with mRNA capture and reverse transcription will enable whole transcriptome processing at the single-cell level using a single device. Gene expression levels are maintained after amplification, a key feature that will lead to greater acceptance in the biological community. REFERENCES [1] R. van Gelder, M. von Zastrow, A. Yool, W. Dement, J. Barchas and J. Eberwine, PNAS, 87, pp. 1663-1667, (1990). [2] B. Satterfield, S. Stern, M. Caplan, K. Hukari and J. West, Analytical Chemistry, 79, pp. 6230-6235, (2007). [3] G. Jiang and D. Harrison, The Analyst, 125, pp. 2176-2179, (2000). [4] J. Zhong, Y. Chen, J. Marcus, A. Scherer, S. Quake, C. Taylor and L. Weiner, Lab on a Chip, 8, pp. 68-74, (2008). [5] N. Bontoux, L. Dauphinot, T. Vitalis, V. Studer, Y. Chen, J. Rossier and M.-C. Potier, Lab on a Chip, 8, pp. 443-450, (2008). [6] J. S. Marcus, W. F. Anderson and S. R. Quake, Analytical Chemistry, 78, pp. 3084-3089, (2006). [7] J. Kralj, A. Player, D. Petersen, S. Forry, M. Munson, E. Kawasaki and L. Locascio, Micro Total Analysis Systems 2007, Paris, (2007). Twelfth International Conference on Miniaturized Systems for Chemistry and Life Sciences October 12 - 16, 2008, San Diego, California, USA

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