Highly multiplexed single-cell quantitative PCR

<div><p>We present a microfluidic device for rapid gene expression profiling in single cells using multiplexed quantitative polymerase chain reaction (qPCR). This device integrates all processing steps, including cell isolation and lysis, complementary DNA synthesis, pre-amplification, sample splitting, and measurement in twenty separate qPCR reactions. Each of these steps is performed in parallel on up to 200 single cells per run. Experiments performed on dilutions of purified RNA establish assay linearity over a dynamic range of at least 10⁴, a qPCR precision of 15%, and detection sensitivity down to a single cDNA molecule. We demonstrate the application of our device for rapid profiling of microRNA expression in single cells. Measurements performed on a panel of twenty miRNAs in two types of cells revealed clear cell-to-cell heterogeneity, with evidence of spontaneous differentiation manifested as distinct expression signatures. Highly multiplexed microfluidic RT-qPCR fills a gap in current capabilities for single-cell analysis, providing a rapid and cost-effective approach for profiling panels of marker genes, thereby complementing single-cell genomics methods that are best suited for global analysis and discovery. We expect this approach to enable new studies requiring fast, cost-effective, and precise measurements across hundreds of single cells.</p></div

Multiplex RT-qPCR device schematic and operation.

<p>(A) Two cell-processing units. Components include (i) a reagent injection bus, (ii) a 0.3-nL cell capture chamber, (iii) a 10-nL reverse transcription (RT) chamber, (iv) a 50-nL pre-amplification chamber, (v) twenty 0.15-nL sample-splitting chambers, (vi) twenty shared assay-delivery chambers, and (vii) twenty 6.4-nL detection chambers. The “flow” layer is made up of features of six different heights. Control valves and assay-delivery channels are on the same 25 μm high SU8 “control” layer. Assay delivery from the control layer to the flow layer occurs through laser-ablated interlayer connections. Scale bar 0.5 mm. (B) Optical micrograph of a single K562 cell (indicated by a black arrow) caught in a cell trap. Scale bar 50 μm. (C) Optical micrograph of a subsection of the detection array. Control valves are coloured red. Scale bar 0.5 mm. (D-G) Schematic illustration of device operation. (D) Single cells are first loaded into the device, then washed, isolated, and lysed <i>in situ</i>. (E) Reverse transcription brew is then injected into the RT chamber, mixed with the lysate, and then the device is thermocycled. (F) Similarly, multiplexed pre-amplification mix is injected into the device, mixed with cDNA, and then the device is again thermocycled. (G) Finally, the pre-amplification product is split between detection chambers for qPCR.</p

Single cell mRNA expression.

<p>(A) Heatmap of expression values of six mRNA on 175 single K562 cells and 4 no cell controls (NCC). Each column represents a cell, and each row represents an assay. Replicate measurements are grouped according to assay. As expected, murine <i>Gapdh</i> is not detected, only spuriously generating signal in 4/525 detection chambers. No-cell controls are clearly distinguishable from those from single cells, with an average difference in magnitude greater than 100×. The variability between replicate measurements for each gene is much smaller than the variance in expression seen between different cells. (B) Distribution of the copy number of each gene measured in each single cell, along with the mean expression values obtained from previously published single-cell qPCR measurements [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191601#pone.0191601.ref010" target="_blank">10</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191601#pone.0191601.ref012" target="_blank">12</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191601#pone.0191601.ref014" target="_blank">14</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191601#pone.0191601.ref030" target="_blank">30</a>]. Error bars represent the mean ± standard deviation.</p