SINGLE-CELL REAL-TIME PCR: DIRECT PROCESS FROM CELLS TO
DATA
Xu Shi*, Liang-I Lin, Szu-yu Chen, Weimin Gao, Shih-hui Chao, Weiwen Zhang,
and Deirdre R. Meldrum
Center for Biosignatures Discovery Automation, the Biodesign Institute, Arizona State University, USA
ABSTRACT
This paper presents an easily produced, cost-efficient chip-level device which has the ability to perform real-time PCR
with single copy sensitivity. In order to integrate the high sensitivity and accuracy of real-time PCR and advantages of a
miniaturized device, a simple chip-level device is built. We used this device to perform real-time PCR with single copy
sensitivity. The minimum off-chip work will decrease the handling and human error during the whole process which will
help to increase the successful rate and sensitivity of the chip.
KEYWORDS: Single-cell, droplet real-time PCR, single-copy sensitivity
INTRODUCTION
Real-time PCR at the single bacterial cell level is a powerful tool to quantitatively reveal the heterogeneity of cells.
Conventional PCR platforms that utilize microtiter plates or PCR tubes have been widely used, but their large reaction
volumes cannot easily produce sensitive single-cell analysis. Microfluidic devices provide high density, low volume PCR
chambers, but they are usually expensive and require dedicated equipment to manipulate liquid and perform detection. In
order to overcome these limitations, we developed an inexpensive chip-level device that is compatible with a commercial
real-time PCR thermal cycler to perform quantitative PCR for single bacterial cells.
The chip-level device contains an array of stationary, surface-adhering droplets immersed in mineral oil as real-time PCR
chambers. Mineral oil is used to isolate the droplets and prevent evaporation during thermal cycling. The dimensions and
locations of the droplets are controlled by hydrophilic patterning on the cover slip surface. The volume of each chamber is 5
µL on the proof-of-concept version, but can be smaller and the density of the droplets can be higher in the future. Real-time
PCR is performed on a commercially available ABI StepOne real-time PCR thermal cycler.
EXPERIMENTAL
Twelve surface-adhering droplets were defined by hydrophilic patterning on a glass cover slip [1]. They served as realtime PCR reaction chambers when they were immersed in oil (Figure 1). The hydrophilic pattern that confines the aqueous
droplets is the most important process of making this device. In this study, we generated such patterns using Microscale
Plasma Activated Templating (μPLAT) [2], a technique that employs a stencil to expose air plasma only to designed areas to
increase the hydrophilicity of the surface (Figure 2). All of these processes can be easily accomplished and all of the material
can be easily accessed in conventional biological laboratory without special instruments or trained expertise which make it a
low cost device. Due to indirect contact between the droplet and heating block, real temperature in the droplet needs to be
compensated by adjusting the set temperatures of the heating block. A calibration was performed by inserting a 0.076 mmdiameter K-type thermocouple (5SC-TT-K-40-36, OMEGA) into the center of a droplet to empirically adjust the set
temperatures and corresponding durations of the thermal cycler to fit the actual temperatures in the droplets.
Figure 1: (a) The chip contains an array of surfaceadhering droplets submerged in oil. (b) The cross
section view of the chip placed on a thermal cycler,
showing that the droplets are aligned with the wells
of the heating block of the thermal cycler (not to
scale).
978-0-9798064-4-5/µTAS 2011/$20©11CBMS-0001
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15th International Conference on
Miniaturized Systems for Chemistry and Life Sciences
October 2-6, 2011, Seattle, Washington, USA
Figure 2: Chip fabrication process
RESULTS AND DISCUSSION
One of the most important advantages of our device is that it requires no off-chip DNA extraction/purification which is
always required for conventional analysis methods. Pre-mixing and loading the PCR reagent using a pipette are the only offchip work. The streamlined process decreases the bias introduced by human handling and unexpected contamination.
Synechocystis PCC 6803 bacterium was used in this study. The rbcl gene was analyzed to validate the feasibility of our
device. The result proved that our streamlined droplet PCR could successfully amplify the rbcl gene under two different
template concentration levels. The average Cq of each concentration level was 31.35 and 34.56 (Figure 3). The Cq difference
between them is 3.2 for rbcl gene, while under the ideal PCR efficiency the difference should be around 3.3 (i.e. log210). The
results proved that our streamlined droplet device not only could perform real-time PCR, but also the efficiency of our device
almost reaches ideal efficiency.
Figure 3: Template concentrations and Cq values for on-chip PCR experiments
Besides the feasibility of the device, the sensitivity of the chip is another important parameter that needs to be examined.
In order to test the detection limits of our device, we performed a series dilution from ten million cells to digital cell level,
with detection of the 16S rRNA gene. The Cq values of each concentration level were 17.07, 23.19, 28.12, 30.42, 33.2
respectively (Figure 4). The difference between the two highest concentration levels, 107 and 105, was 6.12 and the ideal
condition should be around 6.4 which indicated our chip does reach the ideal condition for different targets. The differences
between other concentrations were all close to the ideal condition. The gap between experimental results and ideal conditions
may be due to inaccurate series dilution under low concentration levels. Based on the series dilution results, our device had
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already reached single digital copy level, but the proof was not solid because of low accuracy of series dilution under such
low concentration level. With the help of a micromanipulator, we directly loaded single bacterial cells on our device. The
real-time PCR still worked with a Cq value of 34.01 and a standard deviation of 1.05 (Figure 4, 5). The results proved that our
device can reach single cell level and have a very good reproducibility at single cell level. Considering that there are only 2
copies of the 16S rRNA gene in one cell, we can make a solid conclusion that our device can reach single copy sensitivity.
When compared with other single copy sensitivity devices which use sophisticated microfluidic devices to achieve the single
copy sensitivity, [3, 4] ours is much simpler and more cost-efficient. The elimination of on-chip liquid handling and
manipulation makes the whole device more straightforward. Although the throughput of the device under this version is
relatively low, in the future, the “personalized” thermal cycler will allow higher throughput which will be helpful for fast
single cell analysis.
Figure 4: Real-time PCR result at 107, 105,
103, 102, 10 and single cell levels.
Figure 5: Amplification curve of a single cell
CONCLUSION
In this study, a new design of an easily fabricated multi-chamber real-time PCR chip was demonstrated. The chip was
cost-efficient when compared with some complicated microfluidic devices [3, 5] and the real-time PCR efficiency followed
the theoretic estimation. The reproducibility from the result indicates that our device is very robust. With the help of the new
device, we successfully extend real-time PCR analysis of a gene target to the single-copy level which is extremely helpful for
single cell analysis where the analytes are always extremely low. The application of this device in biological laboratories will
provide the easy and convenient tools to perform single cell analysis and reveal heterogeneity among cells.
ACKNOWLEDGEMENTS
We gratefully acknowledge the support of this research by the NIH National Human Genome Research Institute, Grant
Number 1 R01HG01497 Microscale Instrument Development for Genomic Analysis and ASU.
REFERENCES
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Xu Shi, Liang-I Lin, Szu-yu Chen, Shih-hui Chao, Weiwen Zhang, and Deirdre R. Meldrum, Real-time PCR of
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[2]
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[3]
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N. R. Beer, B. J. Hindson, E. K. Wheeler, S. B. Hall, K. A. Rose, I. M. Kennedy, and B. W. Colston, On-chip, realtime, single-copy polymerase chain reaction in picoliter droplets, Anal. Chem, 79(22): pp. 8471-8475, 2007
[5]
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Nanoliter-Volume Multiplex TaqMan Polymerase Chain Reaction from A Single Copy Based on Counting Fluorescence
Released Microchambers, Anal. Chem, 76(21): pp. 6434-6439, 2004
CONTACT
*Xu Shi, email: xu.shi@asu.edu
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