Difference between revisions of "Team:NUDT CHINA/Description"

Line 384: Line 384:
 
       <div style="margin-top:80px;" id="content-wrap-ink"><!-- adjust in add_JS -->
 
       <div style="margin-top:80px;" id="content-wrap-ink"><!-- adjust in add_JS -->
 
<div class="row footer-link" style="">
 
<div class="row footer-link" style="">
 
  
  
 
<h2>
 
<h2>
<span>Abstract</span>
+
<span><span style="color:#7f1015">Abstract</span></span><hr />
 
</h2>
 
</h2>
<p>
+
 
<b><span style="font-size:18px;">&nbsp;</span></b>
+
<p style="text-indent:22pt;">
</p>
+
<span style="line-height:2;font-family:Perpetua;font-size:18px;">MicroRNAs,
<p>
+
serve as critical gene expression regulators at the transcriptional and
<span style="font-size:18px;">MicroRNAs, serve as critical gene
+
post-transcriptional levels, have also been found as important blood-based
expression regulators at the transcriptional and post-transcriptional levels,
+
biomarkers for early detection of cancers. However, their current in vitro
have also been found as important blood-based biomarkers for early detection of
+
detection methods are relatively complex, costly and low sensitive. Our project
cancers. However, their current in vitro detection methods are relatively
+
attempts to establish a novel in vitro microRNA detection system which is
complex, costly and low sensitive. Our project attempts to establish a novel in
+
rapid, efficient, sensitive and specific. In this system, CRISPR-Cas9 technique
vitro microRNA detection system which is rapid, efficient, sensitive and
+
is modified to integrate with split-luciferase or split-HRP reporting systems.
specific. In this system, CRISPR-Cas9 technique is modified to integrate with
+
The advanced rolling circle amplification technology and cell-free expression
split-luciferase or split-HRP reporting systems. The advanced rolling circle
+
system are also involved and optimized. This system may ideally be compatible
amplification technology and cell-free expression system are also involved and
+
for the detection of various series of small non-coding RNAs. To our knowledge,
optimized. This system may ideally be compatible for the detection of various
+
we are the first to use the CRISPR-Cas9 system as a small non-coding RNA
series of small non-coding RNAs. To our knowledge, we are the first to use the
+
monitor in vitro. Its establishment and further development might provide a new
CRISPR-Cas9 system as a small non-coding RNA monitor in vitro. Its
+
approach for rapid and low-cost cancer screening, virus detection and curative
establishment and further development might provide a new approach for rapid
+
efficacy assessment.</span>  
and low-cost cancer screening, virus detection and curative efficacy
+
assessment.</span>
+
</p>
+
<p>
+
<span style="font-size:18px;">&nbsp;</span>
+
 
</p>
 
</p>
 +
</br>
 
<h2>
 
<h2>
<span>Introduction</span>
+
<span><span style="color:#7f1015">Introduction</span></span><hr />
 
</h2>
 
</h2>
<p>
+
<p style="text-indent:22pt;">
<b><span style="font-size:18px;">&nbsp;</span></b>
+
<span style="line-height:2;font-family:Perpetua;font-size:18px;">Nowadays,
 +
cancers, due to their high incidence and serious mortality, are affecting
 +
populations in all countries and all regions (Figure 1). However, in most
 +
countries, resources for prevention and diagnosis of cancer still remain
 +
limited due to their high cost and low cost-effectiveness</span><sup><span style="line-height:2;font-family:Perpetua;font-size:18px;">1</span></sup><span style="line-height:2;font-family:Perpetua;font-size:18px;">, whereas the early
 +
detection of cancer has been proven to result in improved survival, less
 +
extensive treatment and less possibility to metastasis</span><sup><span style="line-height:2;font-family:Perpetua;font-size:18px;">2-4</span></sup><span style="line-height:2;font-family:Perpetua;font-size:18px;">. Such situation
 +
highlighted the undiminished importance of the development of a low-cost,
 +
easily accessible and rapid tool for early screening and detection of cancers.</span>  
 
</p>
 
</p>
<p>
+
<p style="text-indent:22pt;">
<span style="font-size:18px;">Nowadays, cancers, due to their high
+
<span style="line-height:2;font-family:Perpetua;font-size:18px;">&nbsp;</span>  
incidence and serious mortality, are affecting populations in all countries and
+
all regions (Figure 1). However, in most countries, resources for prevention
+
and diagnosis of cancer still remain limited due to their high cost and low
+
cost-effectiveness</span><sup><span style="font-size:18px;">1</span></sup><span style="font-size:18px;">,
+
whereas the early detection of cancer has been proven to result in improved
+
survival, less extensive treatment and less possibility to metastasis</span><sup><span style="font-size:18px;">2-4</span></sup><span style="font-size:18px;">.
+
Such situation highlighted the undiminished importance of the development of a
+
low-cost, easily accessible and rapid tool for early screening and detection of
+
cancers.</span>
+
</p>
+
<p>
+
<span style="font-size:18px;">&nbsp;</span>
+
 
</p>
 
</p>
  
 
</html>
 
</html>
[[File:T--NUDT CHINA--introfig1.jpg|700px|center ]]
+
[[File:T--NUDT CHINA--introfig1.jpg|700px|center]]
 
<html>
 
<html>
 
 
<p>
 
<p>
<b><span style="font-size:14px;">Figure
+
<b><span style="line-height:2;font-family:Perpetua;font-size:18px;">Figure 1.
1. Global distribution of estimated age-standardized world cancer incidence
+
Global distribution of estimated age-standardized world cancer incidence rate
rate (ASR) per 100 000 in (A) men, (B) women, and mortality rate (ASR) per 100
+
(ASR) per 100 000 in (A) men, (B) women, and mortality rate (ASR) per 100 000
000 in (C) men and (D) women. (WHO, World cancer report, 2014)</span></b>
+
in (C) men and (D) women. (WHO, World cancer report, 2014)</span></b>  
 
</p>
 
</p>
<p align="center" style="text-align:center;">
+
<p align="center" style="text-align:center;text-indent:22.1pt;">
<b><span style="font-size:18px;">&nbsp;</span></b>
+
<b><span style="line-height:2;font-family:Perpetua;font-size:18px;">&nbsp;</span></b>  
 
</p>
 
</p>
<p>
+
<p style="text-indent:22pt;">
<span style="font-size:18px;">MicroRNAs (miRNAs), as a kind of small
+
<span style="line-height:2;font-family:Perpetua;font-size:18px;">MicroRNAs
non-coding RNA containing approximately 22 nucleic acids, have been proven to
+
(miRNAs), as a kind of small non-coding RNA containing approximately 22 nucleic
play important roles on post-transcriptional regulation of the gene expression,
+
acids, have been proven to play important roles on post-transcriptional regulation
thus involving in the regulation of many important biological events</span><sup><span style="font-size:18px;">5</span></sup><span style="font-size:18px;">.
+
of the gene expression, thus involving in the regulation of many important
Recently, it was reported that serum miRNAs can serve as a promising cancer
+
biological events</span><sup><span style="line-height:2;font-family:Perpetua;font-size:18px;">5</span></sup><span style="line-height:2;font-family:Perpetua;font-size:18px;">. Recently, it was reported
biomarker because their expression pattern can be correlated with cancer type,
+
that serum miRNAs can serve as a promising cancer biomarker because their
stage, and other clinical variables, which then, implying that miRNA profiling
+
expression pattern can be correlated with cancer type, stage, and other
can be used as a tool for cancer diagnosis and prognosis</span><sup><span style="font-size:18px;">6-8</span></sup><span style="font-size:18px;">.
+
clinical variables, which then, implying that miRNA profiling can be used as a
Moreover, circulating miRNAs have been proven to remain stable under some
+
tool for cancer diagnosis and prognosis</span><sup><span style="line-height:2;font-family:Perpetua;font-size:18px;">6-8</span></sup><span style="line-height:2;font-family:Perpetua;font-size:18px;">. Moreover, circulating miRNAs
extreme condition such as RNase exposure, multiple freeze-thaw cycles, and
+
have been proven to remain stable under some extreme condition such as RNase
extreme pH, thus making them strong candidates for low-cost detection and
+
exposure, multiple freeze-thaw cycles, and extreme pH, thus making them strong
analysis </span><sup><span style="font-size:18px;">9</span></sup><span style="font-size:18px;">.
+
candidates for low-cost detection and analysis </span><sup><span style="line-height:2;font-family:Perpetua;font-size:18px;">9</span></sup><span style="line-height:2;font-family:Perpetua;font-size:18px;">. However, due to their
However, due to their short length, low expression level and high homologous
+
short length, low expression level and high homologous sequence similarity, the
sequence similarity, the quantified detection and analyzation of circulating
+
quantified detection and analyzation of circulating miRNAs remain challenging
miRNAs remain challenging nowadays. Old-schools such as Northern Blotting,
+
nowadays. Old-schools such as Northern Blotting, microarray and qRT-PCR
microarray and qRT-PCR technique are still our approach to detect and analyze
+
technique are still our approach to detect and analyze the quantity of miRNA</span><sup><span style="line-height:2;font-family:Perpetua;font-size:18px;">10</span></sup><span style="line-height:2;font-family:Perpetua;font-size:18px;">. Notably, the expanded
the quantity of miRNA</span><sup><span style="font-size:18px;">10</span></sup><span style="font-size:18px;">.
+
application of these techniques, as well as some other new approaches such as
Notably, the expanded application of these techniques, as well as some other
+
bioluminescence</span><sup><span style="line-height:2;font-family:Perpetua;font-size:18px;">11</span></sup><span style="line-height:2;font-family:Perpetua;font-size:18px;">, Nanopore sensors</span><sup><span style="line-height:2;font-family:Perpetua;font-size:18px;">12</span></sup><span style="line-height:2;font-family:Perpetua;font-size:18px;"> were severely limited
new approaches such as bioluminescence</span><sup><span style="font-size:18px;">11</span></sup><span style="font-size:18px;">,
+
due to their relatively low sensitivity (which were mostly nM sensitivity
Nanopore sensors</span><sup><span style="font-size:18px;">12</span></sup><span style="font-size:18px;"> were
+
severely limited due to their relatively low sensitivity (which were mostly nM sensitivity
+
 
against the pM or even fM concentration of blood miRNA), cumbersome and complex
 
against the pM or even fM concentration of blood miRNA), cumbersome and complex
in operation, and relative high cost. More recently, Deng </span><i><span style="font-size:18px;">et.al </span></i><span style="font-size:18px;">reported a single-molecule resolution </span><i><span style="font-size:18px;">in situ</span></i><span style="font-size:18px;"> miRNA detection technique based on rolling circle
+
in operation, and relative high cost. More recently, Deng </span><i><span style="line-height:2;font-family:Perpetua;font-size:18px;">et.al </span></i><span style="line-height:2;font-family:Perpetua;font-size:18px;">reported a single-molecule resolution </span><i><span style="line-height:2;font-family:Perpetua;font-size:18px;">in situ</span></i><span style="line-height:2;font-family:Perpetua;font-size:18px;"> miRNA detection technique based on rolling circle
amplification (RCA)</span><sup><span style="font-size:18px;">13</span></sup><span style="font-size:18px;">.
+
amplification (RCA)</span><sup><span style="line-height:2;font-family:Perpetua;font-size:18px;">13</span></sup><span style="line-height:2;font-family:Perpetua;font-size:18px;">. However, this approach
However, this approach has been restricted only in the application of cellular </span><i><span style="font-size:18px;">in situ</span></i><span style="font-size:18px;"> analysis. Its expandability to
+
has been restricted only in the application of cellular </span><i><span style="line-height:2;font-family:Perpetua;font-size:18px;">in situ</span></i><span style="line-height:2;font-family:Perpetua;font-size:18px;"> analysis. Its expandability to circulating miRNA detection
circulating miRNA detection still faces a major problem that the degree of one-step
+
still faces a major problem that the degree of one-step signal amplification and
signal amplification and differentiation might not be sufficient to meet the
+
differentiation might not be sufficient to meet the requirements of sensitivity
requirements of sensitivity and specificity. At the main time, such method
+
and specificity. At the main time, such method still relies on equipment such
still relies on equipment such as Fluoresce microplate readers or fluorescence
+
as Fluoresce microplate readers or fluorescence microscopes, which are highly
microscopes, which are highly costly.</span>
+
costly. </span>  
 
</p>
 
</p>
<p>
+
</br>
<span style="font-size:18px;">&nbsp;</span>
+
</p>
+
 
+
 
</html>
 
</html>
[[File:T--NUDT CHINA--introfig2.jpg|700px|center ]]
+
[[File:T--NUDT CHINA--introfig2.jpg|700px|center]]
 
<html>
 
<html>
 
+
</br>
 
<p>
 
<p>
<b><span style="font-size:14px;">Figure
+
<b><span style="line-height:2;font-family:Perpetua;font-size:18px;">Figure 2.
2. Workflow for our CRISPR-based blood-microRNA detection system.</span></b>
+
Workflow for our CRISPR-based blood-microRNA detection system.</span></b>  
 
</p>
 
</p>
 
<p>
 
<p>
<span style="font-size:14px;">Using sequence information from online
+
<span style="line-height:2;font-family:Perpetua;font-size:16px;">Using sequence information from online databases,
databases, probes for RCA reaction were designed in silico. Once synthesized
+
probes for RCA reaction were designed in silico. Once synthesized and sealed to
and sealed to form the dumbbell structure, the probe, together with other
+
form the dumbbell structure, the probe, together with other necessary materials
necessary materials can be embedded into tubes and freeze-dried to remain
+
can be embedded into tubes and freeze-dried to remain stable in room
stable in room temperature for a relatively long time. For the detection
+
temperature for a relatively long time. For the detection process, serum
process, serum samples were pre-treated by boiling in 95℃ for 15 min to expose the
+
samples were pre-treated by boiling in 95</span><span style="line-height:2;font-family:Perpetua;font-size:18px;">℃</span><span style="line-height:2;font-family:Perpetua;font-size:18px;"> for 15 min to expose
miRNAs completely. The amount of the specific RNA was indicated by a color
+
the miRNAs completely. The amount of the specific RNA was indicated by a color
difference in the tube from colorless to blue</span>
+
difference in the tube from colorless to blue</span>  
 
</p>
 
</p>
<p>
+
</br>
<b><span style="font-size:18px;">&nbsp;</span></b>
+
<p style="text-indent:22pt;">
 +
<span style="line-height:2;font-family:Perpetua;font-size:18px;">In
 +
our project, we designed a novel cell-free platform built with synthetic
 +
bio-components to achieve the low-cost, handy and visualized detection of serum
 +
miRNAs, which can be employed in low-resource settings (Figure 2). Using miR
 +
let-7a (a bio-marker for non-small cell lung cancer (NSCLC)) as a demo of our
 +
scheme, we modified the RCA based DNA amplification system and introduced it
 +
into nucleic acid detection in liquid samples such as serum, and then conducted
 +
Sybr I mediated fluorescent assay for its validation and assessment. The
 +
improvements of sensitivity and specificity of RCA output signal as well as the
 +
visualization of RCA outputs were achieved through a single guide RNA (sgRNA)
 +
mediated dCas9 binding system and a conjugated split-HRP reporting system.
 +
Meanwhile, a mathematic model was also developed to provide theoretical
 +
approval to our scheme and basic guideline for wet-lab experiments. Finally, we
 +
employed a simple sample-pretreatment protocol to reliably expose miRNAs in
 +
serum samples and demonstrated robust detection with this scheme to compare
 +
let-7a concentrations among blood samples collected from NSCLC patients and healthy
 +
volunteers. </span>  
 
</p>
 
</p>
<p>
+
<p style="text-indent:22pt;">
<span style="font-size:18px;">In our project, we designed a novel cell-free
+
<span style="line-height:2;font-family:Perpetua;font-size:18px;">&nbsp;</span>  
platform built with synthetic bio-components to achieve the low-cost, handy and
+
visualized detection of serum miRNAs, which can be employed in low-resource
+
settings (Figure 2). Using miR let-7a (a bio-marker for non-small cell lung
+
cancer (NSCLC)) as a demo of our scheme, we modified the RCA based DNA
+
amplification system and introduced it into nucleic acid detection in liquid
+
samples such as serum, and then conducted Sybr I mediated fluorescent assay for
+
its validation and assessment. The improvements of sensitivity and specificity
+
of RCA output signal as well as the visualization of RCA outputs were achieved
+
through a single guide RNA (sgRNA) mediated dCas9 binding system and a
+
conjugated split-HRP reporting system. Meanwhile, a mathematic model was also
+
developed to provide theoretical approval to our scheme and basic guideline for
+
wet-lab experiments. Finally, we employed a simple sample-pretreatment protocol
+
to reliably expose miRNAs in serum samples and demonstrated robust detection
+
with this scheme to compare let-7a concentrations among blood samples collected
+
from NSCLC patients and healthy volunteers.</span>
+
</p>
+
<p>
+
<span style="font-size:18px;">&nbsp;</span>
+
 
</p>
 
</p>
 
<h2>
 
<h2>
<span>Improvements
+
<span><span style="color:#7f1015">Improvement We Made</span></span><hr />
we made</span>
+
 
</h2>
 
</h2>
 
<p>
 
<p>
<b><span style="font-size:18px;">&nbsp;</span></b>
+
<span style="line-height:2;font-family:Perpetua;font-size:18px;"><strong>BBa_K1789003 and BBa_K1789004</strong></span>  
 
</p>
 
</p>
<p>
+
<p style="text-indent:22pt;">
<span style="font-size:18px;"><strong>BBa_K1789003 and BBa_K1789004</strong></span>
+
<span style="line-height:2;font-family:Perpetua;font-size:18px;">In
 +
our project this year, a new protein-protein interaction (PPI) toolkit
 +
containing several split reporting systems were modified and designed and
 +
introduced into the registry. As a classical PPI indicator, split-GFP system,
 +
developed previously in our project in iGEM2015 (BBa_K1789003 and
 +
BBa_K1789004), was also included in our kit. Several improvements has been made
 +
for this system including:</span>  
 
</p>
 
</p>
<p>
+
<p style="text-indent:-0.55pt;">
<span style="font-size:18px;">In our project this year, a new
+
<span style="line-height:2;font-family:Perpetua;font-size:18px;">1.</span><span style="line-height:2;font-family:Perpetua;font-size:18px;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span><span style="line-height:2;font-family:Perpetua;font-size:18px;">Improved
protein-protein interaction (PPI) toolkit containing several split reporting
+
characterization for previous parts</span>  
systems were modified and designed and introduced into the registry. As a
+
classical PPI indicator, split-GFP system, developed previously in our project
+
in iGEM2015 (BBa_K1789003 and BBa_K1789004), was also included in our kit.
+
Several improvements has been made for this system including:</span>
+
 
</p>
 
</p>
<p style="text-indent:-18pt;">
+
<p style="text-indent:22pt;">
<span style="font-size:18px;">1.&nbsp;&nbsp; Improved
+
<span style="line-height:2;font-family:Perpetua;font-size:18px;">To
characterization for previous parts</span>
+
further improve the function of existing parts, we stimulate an </span><i><span style="line-height:2;font-family:Perpetua;font-size:18px;">in vivo</span></i><span style="line-height:2;font-family:Perpetua;font-size:18px;"> PPI situation, and tried to
 +
optimize the culture condition for a better signal-to-noise ratio (SNR). For
 +
such matter, two devices, containing split-GFP fragments and a complete or
 +
spited zinc finger protein, were built under control of a lac operon controlled
 +
T7 promoter. The complete zinc finger protein was to stimulate a PPI positive
 +
situation, while the split one was to stimulate a PPI negative situation
 +
(Figure 3A). Fluorescence signal was detected by a microplate reader after an
 +
overnight culture under various conditions.</span><span style="line-height:2;font-family:Perpetua;font-size:18px;">&nbsp; </span><span style="line-height:2;font-family:Perpetua;font-size:18px;">Relative fluorescence intensity was then calculated with normalization
 +
of OD</span><sub><span style="line-height:2;font-family:Perpetua;font-size:18px;">600</span></sub><span style="line-height:2;font-family:Perpetua;font-size:18px;"> value. The relative fluorescence intensity of each control
 +
group was set arbitrarily at 1.0 (data not shown), and the levels of the other
 +
groups were adjusted correspondingly. Results shown a better SNR under 20</span><span style="line-height:2;font-family:Perpetua;font-size:18px;">℃</span><span style="line-height:2;font-family:Perpetua;font-size:18px;"> and 0.5mM IPTG induction (Figure 3B). Thus indicating that better performance
 +
of such system could be expected under lower culturing temperature.</span>  
 
</p>
 
</p>
<p>
+
<p style="text-indent:22pt;">
<span style="font-size:18px;">To further improve the
+
<span style="line-height:2;font-family:Perpetua;font-size:18px;">&nbsp;</span>  
function of existing parts, we stimulate an </span><i><span style="font-size:18px;">in
+
vivo</span></i><span style="font-size:18px;"> PPI situation, and tried to optimize the culture condition for a
+
better signal-to-noise ratio (SNR). For such matter, two devices, containing
+
split-GFP fragments and a complete or spited zinc finger protein, were built
+
under control of a lac operon controlled T7 promoter. The complete zinc finger
+
protein was to stimulate a PPI positive situation, while the split one was to
+
stimulate a PPI negative situation (Figure 3A). Fluorescence signal was
+
detected by a microplate reader after an overnight culture under various
+
conditions.&nbsp; Relative fluorescence
+
intensity was then calculated with normalization of OD</span><sub><span style="font-size:18px;">600</span></sub><span style="font-size:18px;"> value.</span>
+
 
</p>
 
</p>
 
<p>
 
<p>
<span style="font-size:18px;">Results shown a better
+
<b><span style="line-height:2;font-family:Perpetua;font-size:18px;">Figure
SNR under 20℃ and 0.5mM IPTG induction (Figure 3B). Thus
+
3 The green fluorescence (Ex: 488 nm; Em: 538 nm) of split GFP after overnight
indicating that better performance of such system could be expected under lower
+
culture of E. coli with or without IPTG induction under different express
culturing temperature.</span>
+
condition. </span></b>  
 
</p>
 
</p>
 
<p>
 
<p>
<span style="font-size:18px;">&nbsp;</span>
+
<span style="line-height:2;font-family:Perpetua;font-size:18px;">Relative fluorescence intensity was
 +
calculated with normalization of the OD600 value. The relative fluorescence
 +
intensity of each control group was set arbitrarily at 1.0 (data not shown),
 +
and the levels of the other groups were adjusted correspondingly. This
 +
experiment was run in three parallel reactions, and the data represent results
 +
obtained from at least three independent experiments. *p&lt;0.05, **p&lt;0.01.</span>  
 
</p>
 
</p>
<p align="center" style="text-align:center;">
+
<p style="text-indent:22pt;">
<b><span style="font-size:14px;">(Figure 3)</span></b>
+
<span style="line-height:2;font-family:Perpetua;font-size:18px;">&nbsp;</span>  
 
</p>
 
</p>
<p>
+
<p style="text-indent:22pt;">
<span style="font-size:18px;">&nbsp;</span>
+
<span style="line-height:2;font-family:Perpetua;font-size:18px;">&nbsp;</span>  
</p>
+
<p>
+
<span style="font-size:18px;">&nbsp;</span>
+
 
</p>
 
</p>
<p style="text-indent:-18pt;">
+
<p style="text-indent:6.5pt;">
<span style="font-size:18px;">2.&nbsp;&nbsp; To
+
<span style="line-height:2;font-family:Perpetua;font-size:18px;">2.</span><span style="line-height:2;font-family:Perpetua;font-size:18px;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span><span style="line-height:2;font-family:Perpetua;font-size:18px;">To
 
further improve the function of split-GFP system, another method of splitting
 
further improve the function of split-GFP system, another method of splitting
 
GFP was introduced and tested in our project. Instead of a traditional two-part
 
GFP was introduced and tested in our project. Instead of a traditional two-part
 
split, we split the GFP protein into three fragments namely GFP10 (residues
 
split, we split the GFP protein into three fragments namely GFP10 (residues
194-212), GFP11 (residues 213-233) and GFP 1-9 (residues 1-193) [sci-reps 10.103/srep02854]. Due to their short
+
194-212), GFP11 (residues 213-233) and GFP 1-9 (residues 1-193) </span><span style="line-height:2;font-family:Perpetua;font-size:18px;">[sci-reps 10.103/srep02854]</span><span style="line-height:2;font-family:Perpetua;font-size:18px;">. Due to their short
 
length, two small fragments can be easily fused onto proteins with less
 
length, two small fragments can be easily fused onto proteins with less
affection on their folding (figure 4A).</span>
+
affection on their folding </span><span style="line-height:2;font-family:Perpetua;font-size:18px;">(figure 4A)</span><span style="line-height:2;font-family:Perpetua;font-size:18px;">. </span>  
 
</p>
 
</p>
 
<p>
 
<p>
<span style="font-size:18px;">&nbsp;</span>
+
<span style="line-height:2;font-family:Perpetua;font-size:18px;">&nbsp;</span>  
 
</p>
 
</p>
<p align="center" style="text-align:center;">
+
<p align="center" style="text-align:center;text-indent:22.1pt;">
<b><span style="font-size:14px;">(Figure 4)</span></b>
+
<b><span style="line-height:2;font-family:Perpetua;font-size:18px;">(Figure
 +
4)</span></b>  
 
</p>
 
</p>
<p align="center" style="text-align:center;">
+
<p style="text-indent:22pt;">
<b><span style="font-size:18px;">&nbsp;</span></b>
+
<span style="line-height:2;font-family:Perpetua;font-size:18px;">&nbsp;</span>  
 
</p>
 
</p>
<p>
+
<p style="text-indent:22pt;">
<span style="font-size:18px;">Comparing with previous
+
<span style="line-height:2;font-family:Perpetua;font-size:18px;">Comparing
split-GFP system, higher SNR was reached under the same expression condition,
+
with previous split-GFP system, higher SNR was reached under the same
while the total signal intensity suffered tolerable decrease (Figure 4B).</span>
+
expression condition, while the total signal intensity suffered tolerable
 +
decrease (Figure 4B).</span>  
 
</p>
 
</p>
 
<p>
 
<p>
<span style="font-size:18px;">&nbsp;</span>
+
<span style="line-height:2;font-family:Perpetua;font-size:18px;">&nbsp;</span>  
 
</p>
 
</p>
 
<h2>
 
<h2>
<span>Reference</span>
+
<span><span style="color:#7f1015">Reference</span></span><hr />
 
</h2>
 
</h2>
</br>
 
 
</p>
 
 
<p style="text-indent:-36pt;">
 
<p style="text-indent:-36pt;">
<span style="font-size:18px;">1&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Jeong,
+
<span style="line-height:2;font-family:Perpetua;font-size:18px;">1</span><span style="line-height:2;font-family:Perpetua;font-size:18px;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span><span style="line-height:2;font-family:Perpetua;font-size:18px;">Jeong,
 
K. E. &amp; Cairns, J. A. Review of economic evidence in the prevention and
 
K. E. &amp; Cairns, J. A. Review of economic evidence in the prevention and
early detection of colorectal cancer. </span><i><span style="font-size:18px;">Health
+
early detection of colorectal cancer. </span><i><span style="line-height:2;font-family:Perpetua;font-size:18px;">Health
Econ Rev</span></i> <b><span style="font-size:18px;">3</span></b><span style="font-size:18px;">, 20,
+
Econ Rev</span></i><span> </span><b><span style="line-height:2;font-family:Perpetua;font-size:18px;">3</span></b><span style="line-height:2;font-family:Perpetua;font-size:18px;">, 20,
doi:10.1186/2191-1991-3-20 (2013).</span>
+
doi:10.1186/2191-1991-3-20 (2013).</span>  
 
</p>
 
</p>
 
<p style="text-indent:-36pt;">
 
<p style="text-indent:-36pt;">
<span style="font-size:18px;">2&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Etzioni, R.</span><i><span style="font-size:18px;"> et al.</span></i><span style="font-size:18px;"> The case for early detection. </span><i><span style="font-size:18px;">Nat Rev Cancer</span></i> <b><span style="font-size:18px;">3</span></b><span style="font-size:18px;">,
+
<span style="line-height:2;font-family:Perpetua;font-size:18px;">2</span><span style="line-height:2;font-family:Perpetua;font-size:18px;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span><span style="line-height:2;font-family:Perpetua;font-size:18px;">Etzioni, R.</span><i><span style="line-height:2;font-family:Perpetua;font-size:18px;"> et al.</span></i><span style="line-height:2;font-family:Perpetua;font-size:18px;"> The case for early detection. </span><i><span style="line-height:2;font-family:Perpetua;font-size:18px;">Nat Rev Cancer</span></i><span> </span><b><span style="line-height:2;font-family:Perpetua;font-size:18px;">3</span></b><span style="line-height:2;font-family:Perpetua;font-size:18px;">,
243-252, doi:10.1038/nrc1041 (2003).</span>
+
243-252, doi:10.1038/nrc1041 (2003).</span>  
 
</p>
 
</p>
 
<p style="text-indent:-36pt;">
 
<p style="text-indent:-36pt;">
<span style="font-size:18px;">3&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Wolf, A. M.</span><i><span style="font-size:18px;"> et al.</span></i><span style="font-size:18px;"> American Cancer Society guideline for the early detection
+
<span style="line-height:2;font-family:Perpetua;font-size:18px;">3</span><span style="line-height:2;font-family:Perpetua;font-size:18px;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span><span style="line-height:2;font-family:Perpetua;font-size:18px;">Wolf, A. M.</span><i><span style="line-height:2;font-family:Perpetua;font-size:18px;"> et al.</span></i><span style="line-height:2;font-family:Perpetua;font-size:18px;"> American Cancer Society guideline for the early detection
of prostate cancer: update 2010. </span><i><span style="font-size:18px;">CA
+
of prostate cancer: update 2010. </span><i><span style="line-height:2;font-family:Perpetua;font-size:18px;">CA
Cancer J Clin</span></i> <b><span style="font-size:18px;">60</span></b><span style="font-size:18px;">, 70-98,
+
Cancer J Clin</span></i><span> </span><b><span style="line-height:2;font-family:Perpetua;font-size:18px;">60</span></b><span style="line-height:2;font-family:Perpetua;font-size:18px;">, 70-98,
doi:10.3322/caac.20066 (2010).</span>
+
doi:10.3322/caac.20066 (2010).</span>  
 
</p>
 
</p>
 
<p style="text-indent:-36pt;">
 
<p style="text-indent:-36pt;">
<span style="font-size:18px;">4&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; McPhail, S., Johnson, S., Greenberg, D.,
+
<span style="line-height:2;font-family:Perpetua;font-size:18px;">4</span><span style="line-height:2;font-family:Perpetua;font-size:18px;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span><span style="line-height:2;font-family:Perpetua;font-size:18px;">McPhail, S., Johnson, S., Greenberg,
Peake, M. &amp; Rous, B. Stage at diagnosis and early mortality from cancer in
+
D., Peake, M. &amp; Rous, B. Stage at diagnosis and early mortality from cancer
England. </span><i><span style="font-size:18px;">Br J Cancer</span></i> <b><span style="font-size:18px;">112 Suppl 1</span></b><span style="font-size:18px;">, S108-115,
+
in England. </span><i><span style="line-height:2;font-family:Perpetua;font-size:18px;">Br J Cancer</span></i><span> </span><b><span style="line-height:2;font-family:Perpetua;font-size:18px;">112 Suppl 1</span></b><span style="line-height:2;font-family:Perpetua;font-size:18px;">, S108-115,
doi:10.1038/bjc.2015.49 (2015).</span>
+
doi:10.1038/bjc.2015.49 (2015).</span>  
 
</p>
 
</p>
 
<p style="text-indent:-36pt;">
 
<p style="text-indent:-36pt;">
<span style="font-size:18px;">5&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Ambros, V. MicroRNA pathways in flies and
+
<span style="line-height:2;font-family:Perpetua;font-size:18px;">5</span><span style="line-height:2;font-family:Perpetua;font-size:18px;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span><span style="line-height:2;font-family:Perpetua;font-size:18px;">Ambros, V. MicroRNA pathways in flies
worms: growth, death, fat, stress, and timing. </span><i><span style="font-size:18px;">Cell</span></i> <b><span style="font-size:18px;">113</span></b><span style="font-size:18px;">, 673-676
+
and worms: growth, death, fat, stress, and timing. </span><i><span style="line-height:2;font-family:Perpetua;font-size:18px;">Cell</span></i><span> </span><b><span style="line-height:2;font-family:Perpetua;font-size:18px;">113</span></b><span style="line-height:2;font-family:Perpetua;font-size:18px;">, 673-676
(2003).</span>
+
(2003).</span>  
 
</p>
 
</p>
 
<p style="text-indent:-36pt;">
 
<p style="text-indent:-36pt;">
<span style="font-size:18px;">6&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Lu, J.</span><i><span style="font-size:18px;"> et al.</span></i><span style="font-size:18px;"> MicroRNA expression profiles classify human cancers. </span><i><span style="font-size:18px;">Nature</span></i> <b><span style="font-size:18px;">435</span></b><span style="font-size:18px;">, 834-838, doi:10.1038/nature03702 (2005).</span>
+
<span style="line-height:2;font-family:Perpetua;font-size:18px;">6</span><span style="line-height:2;font-family:Perpetua;font-size:18px;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span><span style="line-height:2;font-family:Perpetua;font-size:18px;">Lu, J.</span><i><span style="line-height:2;font-family:Perpetua;font-size:18px;"> et al.</span></i><span style="line-height:2;font-family:Perpetua;font-size:18px;"> MicroRNA expression profiles classify human cancers. </span><i><span style="line-height:2;font-family:Perpetua;font-size:18px;">Nature</span></i><span> </span><b><span style="line-height:2;font-family:Perpetua;font-size:18px;">435</span></b><span style="line-height:2;font-family:Perpetua;font-size:18px;">, 834-838, doi:10.1038/nature03702 (2005).</span>  
 
</p>
 
</p>
 
<p style="text-indent:-36pt;">
 
<p style="text-indent:-36pt;">
<span style="font-size:18px;">7&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Jansson, M. D. &amp; Lund, A. H. MicroRNA
+
<span style="line-height:2;font-family:Perpetua;font-size:18px;">7</span><span style="line-height:2;font-family:Perpetua;font-size:18px;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span><span style="line-height:2;font-family:Perpetua;font-size:18px;">Jansson, M. D. &amp; Lund, A. H.
and cancer. </span><i><span style="font-size:18px;">Mol Oncol</span></i> <b><span style="font-size:18px;">6</span></b><span style="font-size:18px;">, 590-610,
+
MicroRNA and cancer. </span><i><span style="line-height:2;font-family:Perpetua;font-size:18px;">Mol Oncol</span></i><span> </span><b><span style="line-height:2;font-family:Perpetua;font-size:18px;">6</span></b><span style="line-height:2;font-family:Perpetua;font-size:18px;">, 590-610,
doi:10.1016/j.molonc.2012.09.006 (2012).</span>
+
doi:10.1016/j.molonc.2012.09.006 (2012).</span>  
 
</p>
 
</p>
 
<p style="text-indent:-36pt;">
 
<p style="text-indent:-36pt;">
<span style="font-size:18px;">8&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Schultz, N. A.</span><i><span style="font-size:18px;"> et al.</span></i><span style="font-size:18px;"> MicroRNA biomarkers in whole blood for detection of
+
<span style="line-height:2;font-family:Perpetua;font-size:18px;">8</span><span style="line-height:2;font-family:Perpetua;font-size:18px;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span><span style="line-height:2;font-family:Perpetua;font-size:18px;">Schultz, N. A.</span><i><span style="line-height:2;font-family:Perpetua;font-size:18px;"> et al.</span></i><span style="line-height:2;font-family:Perpetua;font-size:18px;"> MicroRNA biomarkers in whole blood for detection of
pancreatic cancer. </span><i><span style="font-size:18px;">JAMA</span></i> <b><span style="font-size:18px;">311</span></b><span style="font-size:18px;">, 392-404,
+
pancreatic cancer. </span><i><span style="line-height:2;font-family:Perpetua;font-size:18px;">JAMA</span></i><span> </span><b><span style="line-height:2;font-family:Perpetua;font-size:18px;">311</span></b><span style="line-height:2;font-family:Perpetua;font-size:18px;">, 392-404,
doi:10.1001/jama.2013.284664 (2014).</span>
+
doi:10.1001/jama.2013.284664 (2014).</span>  
 
</p>
 
</p>
 
<p style="text-indent:-36pt;">
 
<p style="text-indent:-36pt;">
<span style="font-size:18px;">9&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Chen, X.</span><i><span style="font-size:18px;"> et al.</span></i><span style="font-size:18px;"> Characterization of microRNAs in serum: a novel class of
+
<span style="line-height:2;font-family:Perpetua;font-size:18px;">9</span><span style="line-height:2;font-family:Perpetua;font-size:18px;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span><span style="line-height:2;font-family:Perpetua;font-size:18px;">Chen, X.</span><i><span style="line-height:2;font-family:Perpetua;font-size:18px;"> et al.</span></i><span style="line-height:2;font-family:Perpetua;font-size:18px;"> Characterization of microRNAs in serum: a novel class of
biomarkers for diagnosis of cancer and other diseases. </span><i><span style="font-size:18px;">Cell Res</span></i> <b><span style="font-size:18px;">18</span></b><span style="font-size:18px;">, 997-1006,
+
biomarkers for diagnosis of cancer and other diseases. </span><i><span style="line-height:2;font-family:Perpetua;font-size:18px;">Cell Res</span></i><span> </span><b><span style="line-height:2;font-family:Perpetua;font-size:18px;">18</span></b><span style="line-height:2;font-family:Perpetua;font-size:18px;">, 997-1006,
doi:10.1038/cr.2008.282 (2008).</span>
+
doi:10.1038/cr.2008.282 (2008).</span>  
 
</p>
 
</p>
 
<p style="text-indent:-36pt;">
 
<p style="text-indent:-36pt;">
<span style="font-size:18px;">10&nbsp;&nbsp;&nbsp; Hunt, E. A., Broyles, D., Head, T. &amp;
+
<span style="line-height:2;font-family:Perpetua;font-size:18px;">10</span><span style="line-height:2;font-family:Perpetua;font-size:18px;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span><span style="line-height:2;font-family:Perpetua;font-size:18px;">Hunt, E. A., Broyles, D., Head, T. &amp;
Deo, S. K. MicroRNA Detection: Current Technology and Research Strategies. </span><i><span style="font-size:18px;">Annu Rev Anal Chem (Palo Alto Calif)</span></i> <b><span style="font-size:18px;">8</span></b><span style="font-size:18px;">, 217-237, doi:10.1146/annurev-anchem-071114-040343
+
Deo, S. K. MicroRNA Detection: Current Technology and Research Strategies. </span><i><span style="line-height:2;font-family:Perpetua;font-size:18px;">Annu Rev Anal Chem (Palo Alto Calif)</span></i><span> </span><b><span style="line-height:2;font-family:Perpetua;font-size:18px;">8</span></b><span style="line-height:2;font-family:Perpetua;font-size:18px;">, 217-237,
(2015).</span>
+
doi:10.1146/annurev-anchem-071114-040343 (2015).</span>  
 
</p>
 
</p>
 
<p style="text-indent:-36pt;">
 
<p style="text-indent:-36pt;">
<span style="font-size:18px;">11&nbsp;&nbsp;&nbsp; Cissell, K. A., Rahimi, Y., Shrestha, S.,
+
<span style="line-height:2;font-family:Perpetua;font-size:18px;">11</span><span style="line-height:2;font-family:Perpetua;font-size:18px;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span><span style="line-height:2;font-family:Perpetua;font-size:18px;">Cissell, K. A., Rahimi, Y., Shrestha,
Hunt, E. A. &amp; Deo, S. K. Bioluminescence-based detection of microRNA, miR21
+
S., Hunt, E. A. &amp; Deo, S. K. Bioluminescence-based detection of microRNA,
in breast cancer cells. </span><i><span style="font-size:18px;">Anal Chem</span></i> <b><span style="font-size:18px;">80</span></b><span style="font-size:18px;">, 2319-2325, doi:10.1021/ac702577a
+
miR21 in breast cancer cells. </span><i><span style="line-height:2;font-family:Perpetua;font-size:18px;">Anal Chem</span></i><span> </span><b><span style="line-height:2;font-family:Perpetua;font-size:18px;">80</span></b><span style="line-height:2;font-family:Perpetua;font-size:18px;">, 2319-2325, doi:10.1021/ac702577a
(2008).</span>
+
(2008).</span>  
 
</p>
 
</p>
 
<p style="text-indent:-36pt;">
 
<p style="text-indent:-36pt;">
<span style="font-size:18px;">12&nbsp;&nbsp;&nbsp; Venkatesan, B. M. &amp; Bashir, R. Nanopore
+
<span style="line-height:2;font-family:Perpetua;font-size:18px;">12</span><span style="line-height:2;font-family:Perpetua;font-size:18px;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span><span style="line-height:2;font-family:Perpetua;font-size:18px;">Venkatesan, B. M. &amp; Bashir, R.
sensors for nucleic acid analysis. </span><i><span style="font-size:18px;">Nature
+
Nanopore sensors for nucleic acid analysis. </span><i><span style="line-height:2;font-family:Perpetua;font-size:18px;">Nature
nanotechnology</span></i> <b><span style="font-size:18px;">6</span></b><span style="font-size:18px;">, 615-624,
+
nanotechnology</span></i><span> </span><b><span style="line-height:2;font-family:Perpetua;font-size:18px;">6</span></b><span style="line-height:2;font-family:Perpetua;font-size:18px;">, 615-624,
doi:10.1038/nnano.2011.129 (2011).</span>
+
doi:10.1038/nnano.2011.129 (2011).</span>  
 
</p>
 
</p>
 
<p style="text-indent:-36pt;">
 
<p style="text-indent:-36pt;">
<span style="font-size:18px;">13&nbsp;&nbsp;&nbsp; Deng, R.</span><i><span style="font-size:18px;"> et al.</span></i><span style="font-size:18px;"> Toehold-initiated rolling circle amplification for visualizing
+
<span style="line-height:2;font-family:Perpetua;font-size:18px;">13</span><span style="line-height:2;font-family:Perpetua;font-size:18px;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span><span style="line-height:2;font-family:Perpetua;font-size:18px;">Deng, R.</span><i><span style="line-height:2;font-family:Perpetua;font-size:18px;"> et al.</span></i><span style="line-height:2;font-family:Perpetua;font-size:18px;"> Toehold-initiated rolling circle amplification for
individual microRNAs in situ in single cells. </span><i><span style="font-size:18px;">Angew Chem Int Ed Engl</span></i> <b><span style="font-size:18px;">53</span></b><span style="font-size:18px;">,
+
visualizing individual microRNAs in situ in single cells. </span><i><span style="line-height:2;font-family:Perpetua;font-size:18px;">Angew Chem Int Ed Engl</span></i><span> </span><b><span style="line-height:2;font-family:Perpetua;font-size:18px;">53</span></b><span style="line-height:2;font-family:Perpetua;font-size:18px;">,
2389-2393, doi:10.1002/anie.201309388 (2014).</span>
+
2389-2393, doi:10.1002/anie.201309388 (2014).</span>  
 
</p>
 
</p>
 
<p>
 
<p>
<span style="font-size:18px;">&nbsp;</span>
+
<span style="line-height:2;font-family:Perpetua;font-size:18px;">&nbsp;</span>  
 
</p>
 
</p>
 
  
  

Revision as of 02:21, 14 October 2016

NUDT_CHINA 2016