A SINGLE CELL NUCLEUS ARRAY TO MONITOR MESSENGER-RNA
MOLECULE TRANSPORTATION THROUGH NUCLEAR MEMBRANE
Noritada Kaji1-3*, Ryo Koyama1, Takao Yasui1,2,
Tetsuya Higashiyama4-6 and Yoshinobu Baba1,2,7
1
Department of Applied Chemistry, Graduate School of Engineering, Nagoya University, JAPAN
2
ImPACT Research Center for Advanced Nanobiodevices, Nagoya University, JAPAN
3
ERATO Higashiyama Live-Holonics Project, Graduate School of Science, Nagoya University,
JAPAN
4
JST, ERATO, Higashiyama Live-Holonics Project, Nagoya University, JAPAN
5
Division of Biological Science, Graduate School of Science, Nagoya University, JAPAN
6
Institute of Transformative Bio-Molecules, Nagoya University, JAPAN
7
Health Research Institute, National Institute of Advanced Industrial Science and Technology
(AIST), JAPAN
ABSTRACT
In this study, a microchamber array device was designed to capture a single cell, lyse a cell membrane,
and isolate a single cell nucleus, and then mRNA in a single cell nucleus were directly quantitated by using highly sensitive In-Stem Molecular Beacons (ISMB). As a result, the expression of three frequently
used house-keeping genes were successfully quantitated to an accuracy of 100 molecules within a single
cell nucleus.
KEYWORDS: Cell Nucleus, Microchamber Array, messenger RNA
INTRODUCTION
Micro- and nanoscale chamber array on a chip becomes a powerful new tool for bioanalysis since it
could stochastically capture cells or biomolecules at a single cell or molecule level with an easy operation [1]. However, to the best of our knowledge, there are no cell nucleus-targeted assay system on a
chip. Almost all of molecular transportation through nuclear membrane is believed to perform by nuclear
pore complex (NPC). However, in non-viral gene delivery methods, transportation efficiency through
NPC is not so high compared to virus systems and it is believed that most of the non-viral vectors are uptaken during nuclear division cycle, mitosis, whereas pathway of cellular uptake into the cytoplasm is
mainly endocytosis [2]. Several models of lipid bilayer mimicking cellular membrane have been developed such as black lipid membrane (BLM), supported lipid bilayers (SLB), and liposomes. However,
there are no models of nuclear membrane which consists of double lipid bilayer membrane.
In this study, a single cell nucleus array was prepared by using microchamber array for molecular
transportation study through nuclear membrane. To demonstrate the biological significance of this nuclear membrane model, mRNA transportation through nuclear membrane was tried to monitor at a single
cell nucleus level.
EXPERIMENTAL
Microchambers with a diameter of 20 μm and a depth of 30 μm, which was designed based on HeLa
cell size and capture efficiency of a single cell, were fabricated on Cycloolefincopolymer (COC) and
Polymethyl methacrylate (PMMA) substrates. After optimizing the capture process, we found that COC
device is more preferable to observe cell nucleus for a long time since PMMA substrate has a maximum
water absorption ratio of 0.3-0.4% by weight and may cause water evaporation during the long period of
observation. In contrast to PMMA substrate, COC substrate has a maximum water absorption ratio of
0.01% by volume and excellent optical property. Therefore the following experiments were performed by
microchamber array devices fabricated on COC substrate.
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19th International Conference on Miniaturized
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October 25-29, 2015, Gyeongju, KOREA
To capture a single nucleus in a
microchamber, two approaches were
performed. The first approach is that
nucleus of HeLa cells were isolated by
commercially available kit (Nuclei EZ
Prep, Sigma-Aldrich) and then seeded on
the microchamber array devices. The
second approach is that a HeLa cell
suspension
was
seeded
on
the
microchamber array devices and allowed
to sedimentation for 15 min. The cells
were randomly sedimented into the Figure 1. (a) A photo and (b) a SEM image of the micromicrochambers or onto the terrace chamber array device. The final goal of this study is to visubetween the microchambers, and then the alize and quantitate molecular transportation to and out of
device was rinsed and flush the excess cell nucleus through nuclear membrane. (c) Workflow of this
cells remained onto the terrace. After experiment. Cell suspension was seeded and allow to settle
replacing
PBS
buffer
in
the onto the microchambers, extra cells were flushed, and then
microchambers by cell lysis buffer cell membrane was chemically lysed and a single cell nucleus
array was set up to the following experiments.
containing non-ionic detergent and
incubated for 10 min, the solution was
replaced by PBS buffer again and the
extracted nucleus in the microchambers
were observed by confocal microscopy.
To visualize mRNA inside a cell
nucleus, ISMB was designed to hybridize
sequences
of
GlycerAldehyde
3Phosphate DeHydrogenase (GAPDH), βactin, and ubiquitin C. They have 18 to
20 nucleotides sequences to hybridize the
target sequences in the loop region and 6
nucleotides sequences at both ends of the
sequences are hybridized with each other
Figure 2. Visualization of mRNA inside a cell nucleus
and form the stem region. To enhance
captured in the microchamber by ISMB at (a) 100 nM,
S/N ratio, fluorescence dye and quencher
(b) 10 nM, and (c) 1 nM. Histograms of fluorescence
were attached inside the stem region, not
intensity from a single microchamber at (d) 100 nM,
(e) 10 nM, and (f) 1 nM of ISMB. Over 10 nM of
the end of nucleotides such as
ISMB, a single cell nucleus was clearly discriminated
conventional molecular beacon [3]. The
from empty chambers.
performances of these ISMB were
evaluated by fluorescence spectroscopy by adding complementary, non-complementary oligonucleotides
or cell extract. For mRNA detection experiments, the solution containing the ISMB was applied to the
microchamber array devices and incubated for a several minutes. The fluorescence intensities in the
microchambers were detected by EM-CCD camera and analyzed by image analysis software.
RESULTS AND DISCUSSION
As shown in Figure 1b and 1c, microchambers with a diameter of 20 μm and a depth of 30 μm were
fabricated on COC substrate. A single viewing field through fluorescence microscope could capture the
image of 1,458 microchambers. The first approach to capture a single nucleus into a microchamber by
commercially available kit was technically difficult because collected nucleus tend to aggregate each
other and never dispersed even after re-suspended in PBS buffer in a microtube. So we adapted the second approach to isolate a single nuclei into a single microchamber. In this approach, over 70% of the
833
microchambers were occupied by a single HeLa cell and a single cell nuclei was remained over 35% of
the microchambers after the cell lysis treatment. So we could observe about 500 cell nucleus at one time
by microscope. To confirm cell lysis process, HeLa cells were stained by two fluorescent dyes, SYTO16
for nucleus and FM1-34 for cell membrane. Although both of SYTO16 and FM1-34 colors were observed when the HeLa cells were captured in the microchambers, only SYTO16 color was observed after
the cell lysis process.
To visualize mRNA, ISMB was applied to the microchamber array devices. As shown in Figure 2,
mRNA inside a cell nucleus was clearly observed over 10 nM of ISMB. The number of mRNA molecule
of GAPDH gene inside a single cell nucleus was calculated as 4,500 to 7,700 molecules to an accuracy of
100 molecules based on a calibration curve. This value was reasonable compared to the reported value,
from 1,000 to 3,000 molecules in a cell [4].
CONCLUSION
This ISMB and microchamber array system will be a promising technique to quantitate mRNA accurately inside a cell nucleus and monitor molecular transportation through nuclear membrane in a massively parallel manner with an easy operation.
ACKNOWLEDGEMENTS
This work was partly supported by the Nitto Foundation, the Nanotechnology Platform Program
(Molecule and Material Synthesis) of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), the JSPS Grant-in-Aid for Scientific Research (A) 24241050, and the JST under the ERATO Higashiyama Live-Holonics Project.
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CONTACT
*Noritada Kaji; phone: +81-52-789-4498; [email protected]
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