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Result

1. Construction of the plasmids

We constructed five plasmids-pEGFP-miR-21-sponge-6s; pEGFP-miR-21-sponge-2s; pHAGE-pre-miR-21; pSB1C3-miR-21-sponge-2s and pSB1C3-miR-21-sponge-6s. The plasmids of pEGFP-miR-21-sponge-6s and pEGFP-miR-21-sponge-2s were used to monitor the expression of miR-21 in cells. We also used pHAGE-pre-miR-21 to manipulate miR-21 expression in cells. And the plasmids of pSB1C3-miR-21-sponge-6s and pSB1C3-miR-21-sponge-2s were submitted to 2017IGEM (part number: BBa_K2514000 and BBa_K2514001).

1.1 Construction of pEGFP-miR-21-sponge-6s and pEGFP-miR-21-sponge-2s plasmid

1.1.1 Amplification of miR-21-sponge-6s and miR-21-sponge-2s fragment.

To monitor the expression level of miR-21 in different cells, we made two plasmids of miR-21 sponges in the 3’UTR of GFP. Firstly, we designed miR-21 sponges contains six miR-21 binding sites with 3-nt spacers for bulged sites (pEGFP-miR-21-sponge-6s) and miR-21 sponges contains two miR-21 binding sites with 3-nt spacers for bulged sites (pEGFP-miR-21-sponge-2s) based on the sequence of hsa-miR-21 according to the previous study (Ebert MS, Neilson JR, Sharp PA. MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells. Nat Methods. 2007 Sep;4(9):721-6). Then we amplified miR-21-sponge-6s and miR-21-sponge-2s using primers. After that, we purified the PCR products by PCR Purification Kit and digested them with restriction enzymes Xho I and Hind III (Fig 1 A and B).

Fig 1. Electrophoresis of miR-21-sponge-6s (A: lanes 1-3) and miR-21-sponge-2s (B: lanes 4-6) PCR product.

1.1.2 Digested pEGFP-C1 vector

We know that microRNA can bind the 3 'UTR of genes to regulate the expression, so we choose EGFP to be the detector of miR-21. We digested the pEGFP-C1 vectors with restriction enzymes Xho I and Hind III (Fig 2).

Fig 2. Electrophoresis of the digested pEGFP-c1 vector.

1.1.3 Ligation of purified miR-21-sponge-6s and miR-21-sponge-2s fragments to pEGFP-c1 vector

miR-21-sponge-6s fragments and miR-21-sponge-2s fragments were ligated to pEGFP-c1 vector, respectively. Then we selected the positive clones by PCR and sequencing (Fig 3 and 4)

C

Fig 3. Electrophoresis of the PCR identifications of pEGFP- miR-21-sponge-6s (A: lane 1-3) and pEGFP- miR-21-sponge-2s (B: lane 4-6) vector and sequencing map.

Fig 4. Electrophoresis of pEGFP- miR-21-sponge-6s (lane 1,2) and pEGFP- miR-21-sponge-2s (lane 3,4) vector.

1.2 Construction of miR-21- precursor expression plasmid

1.2.1 Amplification of miR-21- precursor fragment.

To set up a model to determent the amount of miR-21 in cells, we made a construct to express miR-21. We designed the specific primers of human miR-21-precursor based on the sequences of miR-21 precursor ( http://www.miRbase.org/cgi-bin/miRna_entry.pl?acc=MI0000077 ) and amplified 72bp fragment by PCR. After that, we digested the purified PCR products with restriction enzymes Sal I and Not I (Fig 5).

Fig 5. Electrophoresis of miR-21- precursor PCR product. The lanes 1-3 showed the miR-21- precursor bands.

1.2.2 Digested pHAGE-puro vector

For eukaryotic expression of has-miR-21 in cells, we digested the eukaryotic expression vector pHAGE-puro with the same restriction enzymes Sal I and Not I (Fig 6).

$Fig 6. Electrophoresis of the digested pHAGE-puro vector.\$

Fig 6. Electrophoresis of the digested pHAGE-puro vector.\

1.2.3 Ligation of purified miR-21-precursor fragment to pHAGE-puro vector

To sequence the miR-21-precursor cloning we got, the purified miR-21-precursor fragment was ligated to pHAGE-puro vector. Then we selected the positive clones by PCR and sequencing (Fig 7 and 8).

A

B

Fig 7. Electrophoresis of the PCR identified production of pHAGE- pre-miR-21 vector (A) and sequencing map (B) .

Fig 8. Electrophoresis of pHAGE-pre-miR-21 vector.

1.3 Construction of pSB-miR-21-sponge-6s and pSB-miR-21-sponge-2s plasmid
1.3.1 Amplification of miR-21-sponge-6s and miR-21-sponge-2s fragment.

In order to submit the parts to IGM2017, we amplified miR-21-sponge-6s and miR-21-sponge-2s using primers. After that, we purified the PCR products by PCR Purification Kit and digested them with restriction enzymes EcoR I and Pst I (Fig 9 A and B).

Fig 9. Electrophoresis of miR-21-sponge-6s (A: lanes 1-3) and miR-21-sponge-2s (B: lanes 4-6) PCR product.

1.3.2 Digested pSB1C3 vector

We digested the pSB1C3 vector with the same restriction enzymes EcoR I and Pst I (Fig 10).

Fig 10. Electrophoresis of the digested pSB1C3 vector.

1.3.3 Ligation of purified miR-21-sponge-6s and miR-21-sponge-2s fragment to pSB1C3 vector

We ligated the purified miR-21-sponge-6s or miR-21-sponge-2s fragment to pSB1C3 vector, respectively. Then we selected the positive clones by PCR and sequencing (Fig 11 and 12).

Fig 12. Electrophoresis of pSB1C3- miR-21-sponge-6s (lane 1, 2) and pSB1C3- miR-21-sponge-2s (lane 3, 4) vector.

1.4 The solid plates of each plasmid constructed (Fig 13)

Fig 13 Solid plates of five plasmids

2. The effect of miR-21 sponges in cells

To detect the validity of our detection device, we transfected pEGFP-miR-21-sponge-6s or pEGFP-miR-21-sponge-2s (0.25ug /well) with pHAGE-puro (0.25ug /well, as negative control) into 293T cells in 24 well-plate, respectively. At same times, we transfected pEGFP-miR-21-sponge-6s or pEGFP-miR-21-sponge-2s (0.25ug/well) with pHAGE-pre-miR-21 (0.25ug/well) into 293T cells. Then the GFP fluorescence was observed under fluorescence microscopy (Fig 14 A,B,C,D). The result suggested our detection device worked very well, and overexpression of miR-21 can decrease the fluorescence intensity of pEGFP-miR-21-sponge-6s and pEGFP-miR-21-sponge-2s.

And we also detected the fluorescence of GFP after transfection pEGFP-miR-21-sponge-6s or pEGFP- miR-21-sponge-2s with or without pHAGE-pre-miR-21 for 24 h by plate reader (SpectraMax i3), and observed that endogenous miR-21 in cells decreased the fluorescence of GFP (Fig 15). The fluorescence of GFP in cells transfect with pEGFP-miR-21-sponge-6s was lower in that of pEGFP-miR-21-sponge-2s, because pEGFP-miR-21-sponge-6s contains six miR-21 binding sites. These results suggested that sponges with six binding sites produced stronger derepressiveeffects than sponges with two binding sites.

Fig 14. The images of 293T cells transfected different plasmids A. pEGFP- miR-21-sponge-6s and pHAGE-puro co-transfected. B. pEGFP-miR-21-sponge-2s and pHAGE-puro co-transfected. C. pEGFP-miR-21-sponge-6s and pHAGE-pre-miR-21 co-transfected. D. pEGFP- miR-21-sponge-2s and pHAGE-pre-miR-21 co-transfected, Bar = 100mm .

Fig 15. The fluorescence of GFP after co-transfection pEGFP-miR-21-sponge-6s or pEGFP- miR-21-sponge-2s with or without pHAGE-pre-miR-21 .

2.1 The effect of miR-21 sponges as a monitor to detect the expression of miR-21

To test the effect of miR-21 sponges as a monitor to detect the expression of miR-21, 293T cells were transfected with pEGFP-miR-21-sponge-6s or pEGFP-miR-21-sponge-2s with different concentration of pHAGE-pre-miR-21. We used the plasmid of pHAGE-pre-miR-21 to quantify miR-21 expression and calculate the copy numbers of miR-21 by using the formula listed below.

copies/ul= (6.02×1023)×(plasmids concentrations ng/ul×10-9)/(DNA length×660)

The standard curve of miR-21-sponge-6s and miR-21-sponge-2s were made by EXCEL, respectively (Fig 16 and 17). We find that when miR-21 copies changed the slope of the standard curve of miR-21-sponge-2s was greater than miR-21-sponge-6s which indicated miR-21-sponge-2s is more sensitive than miR-21-sponge6s. However, the correlation coefficient (R2 value) of miR-21-sponge-6s was 0.9993 and R2 value of miR-21-sponge-6s was 0.8995. The R2 value of miR-21-sponge-6s is better than miR-21-sponge-2s.

Fig 16. The effect of pEGFP-miR-21-sponge-6s or pEGFP-miR-21-sponge-2s on the correlative between fluorescence value of GFP and copy numbers of miR-21.

Fig 17. The standard curve of miR-21-sponge-6s and miR-21-sponge-2s.

Conclusions and perspectives

The results reported here demonstrate that miRNA sponge may be a monitor to detect the expression of miRNA in cells. And we also suggested the slope of the standard curve of miR-21-sponge-2s is better than miR-21-sponge-6s, suggesting the more sensitive of miR-21-sponge-2s as a monitor. Our results for the first time to evaluate the possibility of miRNA sponge for detecting miRNA and provide a new method for diagnosis miRNA in the future. However, there are still lots of issues relative to this new method that we need to consider. Firstly, we should evaluate the sensitivity and specificity of this technique by compare with other method including real-time PCR. Next, whether miRNA sponge contained two binding sites is the best monitor should be considered and tested. More strategies should be thought to improve the sensitivity and specificity of this technique by synthetic biology.

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+										<h1 class="text-align-center">Result</h1>
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-								<h1 class="text-align-center">Result</h1>
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+														<p> <strong>1.&nbsp;&nbsp;&nbsp;&nbsp; Construction of the plasmids</strong>
+														</p>
+														<p>
+															&nbsp; We constructed five plasmids-pEGFP-miR-21-sponge-6s; pEGFP-miR-21-sponge-2s; pHAGE-pre-miR-21; pSB1C3-miR-21-sponge-2s and pSB1C3-miR-21-sponge-6s. The plasmids of pEGFP-miR-21-sponge-6s and pEGFP-miR-21-sponge-2s were used to monitor the expression of miR-21 in cells. We also used pHAGE-pre-miR-21 to manipulate miR-21 expression in cells. And the plasmids of pSB1C3-miR-21-sponge-6s and pSB1C3-miR-21-sponge-2s were submitted to 2017IGEM (part number: BBa_K2514000 and BBa_K2514001).
+														</p>
+														<p> <strong>1.1 Construction of pEGFP-miR-21-sponge-6s and pEGFP-miR-21-sponge-2s plasmid</strong>
+														</p>
+														<p>
+.1.1 Amplification of miR-21-sponge-6s and miR-21-sponge-2s fragment.
+														</p>
+														<p>
+															To monitor the expression level of miR-21 in different cells, we made two plasmids of miR-21 sponges in the 3’UTR of GFP. Firstly, we designed miR-21 sponges contains six miR-21 binding sites with 3-nt spacers for bulged sites (pEGFP-miR-21-sponge-6s) and miR-21 sponges contains two miR-21 binding sites with 3-nt spacers for bulged sites (pEGFP-miR-21-sponge-2s) based on the sequence of hsa-miR-21 according to the previous study (Ebert MS, Neilson JR, Sharp PA. MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells. Nat Methods. 2007 Sep;4(9):721-6). Then we amplified miR-21-sponge-6s and miR-21-sponge-2s using primers. After that, we purified the PCR products by PCR Purification Kit and digested them with restriction enzymes Xho I and Hind III (Fig 1 A and B).
+														</p>
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+																<div style="padding-bottom: 29.0598%; overflow: hidden;" class="image-block-wrapper   has-aspect-ratio" data-description="<p> <em>Fig 1. Electrophoresis of miR-21-sponge-6s (A: lanes 1-3) and miR-21-sponge-2s (B: lanes 4-6) PCR product.</em>
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+																	<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ad39652deaab95256187/1509076299352/"  alt="Fig 1. Electrophoresis of miR-21-sponge-6s (A: lanes 1-3) and miR-21-sponge-2s (B: lanes 4-6) PCR product."  />
+																</noscript>
+																<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ad39652deaab95256187/1509076299352/" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ad39652deaab95256187/1509076299352/" data-image-dimensions="468x136" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f2ad39652deaab95256187" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 1. Electrophoresis of miR-21-sponge-6s (A: lanes 1-3) and miR-21-sponge-2s (B: lanes 4-6) PCR product." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ad39652deaab95256187/1509076299352/?format=500w" data-image-resolution="500w"></div>
+															<div class="image-caption-wrapper">
+																<div class="image-caption">
+																	<p> <em>Fig 1. Electrophoresis of miR-21-sponge-6s (A: lanes 1-3) and miR-21-sponge-2s (B: lanes 4-6) PCR product.</em>
+																	</p>
+																</div>
+															</div>
+														</div>
+													</div>
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+													<p>1.1.2 &nbsp;&nbsp;&nbsp; Digested pEGFP-C1 vector</p>
+													<p>
+														We know that microRNA can bind the 3 'UTR of genes to regulate the expression, so we choose EGFP to be the detector of miR-21. We digested the pEGFP-C1 vectors with restriction enzymes Xho I and Hind III (Fig 2).
+													</p>
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+															<div style="padding-bottom: 49.7409%; overflow: hidden;" class="image-block-wrapper   has-aspect-ratio" data-description="<p>
+																<em>Fig 2. Electrophoresis of the digested pEGFP-c1 vector.</em>
+															</p>
+															" id="yui_3_17_2_1_1509572305608_136">
+															<noscript>
+																<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ad766c3194b973e71691/1509076361545/Fig+2.+Electrophoresis+of+the+digested+pEGFP-c1+vector."  alt="Fig 2. Electrophoresis of the digested pEGFP-c1 vector."  />
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+															<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ad766c3194b973e71691/1509076361545/Fig+2.+Electrophoresis+of+the+digested+pEGFP-c1+vector." data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ad766c3194b973e71691/1509076361545/Fig+2.+Electrophoresis+of+the+digested+pEGFP-c1+vector." data-image-dimensions="193x96" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f2ad766c3194b973e71691" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 2. Electrophoresis of the digested pEGFP-c1 vector." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ad766c3194b973e71691/1509076361545/Fig+2.+Electrophoresis+of+the+digested+pEGFP-c1+vector.?format=300w" data-image-resolution="300w"></div>
+														<div class="image-caption-wrapper">
+															<div class="image-caption">
+																<p>
+																	<em>Fig 2. Electrophoresis of the digested pEGFP-c1 vector.</em>
+																</p>
+															</div>
+														</div>
+													</div>
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+											</div>
+										</div>
+										<div class="sqs-block html-block sqs-block-html" data-block-type="2" id="block-yui_3_17_2_93_1509069313608_69052">
+											<div class="sqs-block-content">
+												<p>
+													<!--[if gte vml 1]>
+													<v:shape
+ id="_x0000_i1026" type="#_x0000_t75" style='width:193pt;height:97pt'>
+														<v:imagedata src="file:////Users/tianyuwang/Library/Group%20Containers/UBF8T346G9.Office/msoclip1/01/clip_image003.png"
+&nbsp; o:title=""/>
+													</v:shape>
+													<![endif]-->
+												</p>
+												<p>
+.1.3 Ligation of purified miR-21-sponge-6s and miR-21-sponge-2s fragments&nbsp; to pEGFP-c1 vector
+												</p>
+												<p>
+													miR-21-sponge-6s fragments and miR-21-sponge-2s fragments were ligated to pEGFP-c1 vector, respectively. Then we selected the positive clones by PCR and sequencing (Fig 3 and 4)
+												</p>
+											</div>
+										</div>
+										<div class="sqs-block image-block sqs-block-image sqs-text-ready" data-block-type="5" id="block-yui_3_17_2_94_1509069313608_11772">
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+													<div class="intrinsic" style="max-width:281.0px;" id="yui_3_17_2_1_1509572305608_154">
+														<div style="padding-bottom: 76.5125%; overflow: hidden;" class="image-block-wrapper   has-aspect-ratio" data-description="" id="yui_3_17_2_1_1509572305608_153">
+															<noscript>
+																<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ae31e4966bac11616540/1509076538529/3.jpg"  alt="3.jpg"  />
+															</noscript>
+															<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ae31e4966bac11616540/1509076538529/3.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ae31e4966bac11616540/1509076538529/3.jpg" data-image-dimensions="281x215" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f2ae31e4966bac11616540" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="3.jpg" src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ae31e4966bac11616540/1509076538529/3.jpg?format=300w" data-image-resolution="300w"></div>
+													</div>
+												</div>
+											</div>
+										</div>
+										<div class="sqs-block image-block sqs-block-image sqs-text-ready" data-block-type="5" id="block-yui_3_17_2_94_1509069313608_17356">
+											<div class="sqs-block-content" id="yui_3_17_2_1_1509572305608_173">
+												<div class="image-block-outer-wrapper layout-caption-below design-layout-inline" id="yui_3_17_2_1_1509572305608_172">
+													<div class="intrinsic" style="max-width:318.0px;" id="yui_3_17_2_1_1509572305608_171">
+														<div style="padding-bottom: 55.9748%; overflow: hidden;" class="image-block-wrapper   has-aspect-ratio" data-description="<p>C</p>
+														<p>
+															<em>
+																Fig 3. Electrophoresis of the PCR identifications of pEGFP- miR-21-sponge-6s (A: lane 1-3) and&amp;nbsp; pEGFP- miR-21-sponge-2s (B: lane 4-6) vector and sequencing map.
+															</em>
+														</p>
+														" id="yui_3_17_2_1_1509572305608_170">
+														<noscript>
+															<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ae499140b7ad7ea3604f/1509076642038/"  alt="C Fig 3. Electrophoresis of the PCR identifications of pEGFP- miR-21-sponge-6s (A: lane 1-3) and  pEGFP- miR-21-sponge-2s (B: lane 4-6) vector and sequencing map."  />
+														</noscript>
+														<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ae499140b7ad7ea3604f/1509076642038/" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ae499140b7ad7ea3604f/1509076642038/" data-image-dimensions="318x178" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f2ae499140b7ad7ea3604f" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="C Fig 3. Electrophoresis of the PCR identifications of pEGFP- miR-21-sponge-6s (A: lane 1-3) and&nbsp; pEGFP- miR-21-sponge-2s (B: lane 4-6) vector and sequencing map." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ae499140b7ad7ea3604f/1509076642038/?format=500w" data-image-resolution="500w"></div>
+													<div class="image-caption-wrapper">
+														<div class="image-caption">
+															<p>C</p>
+															<p>
+																<em>
+																	Fig 3. Electrophoresis of the PCR identifications of pEGFP- miR-21-sponge-6s (A: lane 1-3) and&nbsp; pEGFP- miR-21-sponge-2s (B: lane 4-6) vector and sequencing map.
+																</em>
+															</p>
+														</div>
+													</div>
+												</div>
+											</div>
+										</div>
+									</div>
+									<div class="sqs-block image-block sqs-block-image sqs-text-ready" data-block-type="5" id="block-yui_3_17_2_94_1509069313608_21117">
+										<div class="sqs-block-content" id="yui_3_17_2_1_1509572305608_190">
+											<div class="image-block-outer-wrapper layout-caption-below design-layout-inline" id="yui_3_17_2_1_1509572305608_189">
+												<div class="intrinsic" style="max-width:193.0px;" id="yui_3_17_2_1_1509572305608_188">
+													<div style="padding-bottom: 55.9585%; overflow: hidden;" class="image-block-wrapper   has-aspect-ratio" data-description="<p>
+														<em>
+															Fig 4. Electrophoresis of pEGFP- miR-21-sponge-6s (lane 1,2) and&amp;nbsp; pEGFP- miR-21-sponge-2s (lane 3,4) vector.
+														</em>
+													</p>
+													" id="yui_3_17_2_1_1509572305608_187">
+													<noscript>
+														<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ae59652deaab95257722/1509076660513/"  alt="Fig 4. Electrophoresis of pEGFP- miR-21-sponge-6s (lane 1,2) and  pEGFP- miR-21-sponge-2s (lane 3,4) vector."  />
+													</noscript>
+													<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ae59652deaab95257722/1509076660513/" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ae59652deaab95257722/1509076660513/" data-image-dimensions="193x108" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f2ae59652deaab95257722" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 4. Electrophoresis of pEGFP- miR-21-sponge-6s (lane 1,2) and&nbsp; pEGFP- miR-21-sponge-2s (lane 3,4) vector." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ae59652deaab95257722/1509076660513/?format=300w" data-image-resolution="300w"></div>
+												<div class="image-caption-wrapper">
+													<div class="image-caption">
+														<p>
+															<em>
+																Fig 4. Electrophoresis of pEGFP- miR-21-sponge-6s (lane 1,2) and&nbsp; pEGFP- miR-21-sponge-2s (lane 3,4) vector.
+															</em>
+														</p>
+													</div>
+												</div>
+											</div>
+										</div>
+									</div>
+								</div>
+								<div class="sqs-block html-block sqs-block-html" data-block-type="2" id="block-yui_3_17_2_94_1509069313608_12051">
+									<div class="sqs-block-content">
+										<p>
+											<!--[if gte vml 1]>
+											<v:shape
+ id="_x0000_i1027" type="#_x0000_t75" style='width:281pt;height:214pt'>
+												<v:imagedata src="file:////Users/tianyuwang/Library/Group%20Containers/UBF8T346G9.Office/msoclip1/01/clip_image005.png"
+&nbsp; o:title=""/>
+											</v:shape>
+											<![endif]-->
+										</p>
+										<p>
+											<!--[if gte vml 1]>
+											<v:shape
+ id="_x0000_i1028" type="#_x0000_t75" style='width:318pt;height:178pt'>
+												<v:imagedata src="file:////Users/tianyuwang/Library/Group%20Containers/UBF8T346G9.Office/msoclip1/01/clip_image007.png"
+&nbsp; o:title=""/>
+											</v:shape>
+											<![endif]-->
+										</p>
+										<p>
+											<strong>1.2 &nbsp;&nbsp;&nbsp; Construction of miR-21-</strong>
+											<strong>precursor</strong>
+											<strong>expression plasmid</strong>
+										</p>
+										<p>
+.2.1 &nbsp;&nbsp;&nbsp;&nbsp; Amplification of miR-21- precursor fragment.
+										</p>
+										<p>
+											<strong><!--[if gte vml 1]>
+												<v:shape
+ id="_x0000_i1029" type="#_x0000_t75" style='width:193pt;height:108pt'>
+													<v:imagedata src="file:////Users/tianyuwang/Library/Group%20Containers/UBF8T346G9.Office/msoclip1/01/clip_image009.png"
+&nbsp; o:title=""/>
+												</v:shape>
+												<![endif]--></strong>
+											To set up a model to determent the amount of miR-21 in cells, we made a construct to express miR-21.&nbsp; We designed the specific primers of human miR-21-precursor based on the sequences of miR-21 precursor (
+											<a href="http://www.mirbase.org/cgi-bin/mirna_entry.pl?acc=MI0000077">http://www.miRbase.org/cgi-bin/miRna_entry.pl?acc=MI0000077</a>
+											) and amplified 72bp fragment by PCR. After that, we digested the purified PCR products with restriction enzymes Sal I and Not I (Fig 5).
+										</p>
+									</div>
+								</div>
+								<div class="sqs-block image-block sqs-block-image sqs-text-ready" data-block-type="5" id="block-yui_3_17_2_94_1509069313608_88253">
+									<div class="sqs-block-content" id="yui_3_17_2_1_1509572305608_207">
+										<div class="image-block-outer-wrapper layout-caption-below design-layout-inline" id="yui_3_17_2_1_1509572305608_206">
+											<div class="intrinsic" style="max-width:198.0px;" id="yui_3_17_2_1_1509572305608_205">
+												<div style="padding-bottom: 54.0404%; overflow: hidden;" class="image-block-wrapper   has-aspect-ratio" data-description="<p>
+													<em>Fig 5. Electrophoresis of miR-21-</em>
+													precursor
+													<em>PCR product. The lanes 1-3 showed the miR-21-</em>
+													precursor
+													<em>bands.</em>
+												</p>
+												" id="yui_3_17_2_1_1509572305608_204">
+												<noscript>
+													<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2af200d9297adf936234e/1509076769342/6.jpg"  alt="Fig 5. Electrophoresis of miR-21-precursor PCR product. The lanes 1-3 showed the miR-21-precursor bands."  />
+												</noscript>
+												<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2af200d9297adf936234e/1509076769342/6.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2af200d9297adf936234e/1509076769342/6.jpg" data-image-dimensions="198x107" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f2af200d9297adf936234e" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 5. Electrophoresis of miR-21-precursor PCR product. The lanes 1-3 showed the miR-21-precursor bands." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2af200d9297adf936234e/1509076769342/6.jpg?format=300w" data-image-resolution="300w"></div>
+											<div class="image-caption-wrapper">
+												<div class="image-caption">
+													<p>
+														<em>Fig 5. Electrophoresis of miR-21-</em>
+														precursor
+														<em>PCR product. The lanes 1-3 showed the miR-21-</em>
+														precursor
+														<em>bands.</em>
+													</p>
+												</div>
+											</div>
+										</div>
+									</div>
+								</div>
 							</div>
-							<div class="sqs-block html-block sqs-block-html" data-block-type="2" id="block-77a6b083575b3c254a6d">
+							<div class="sqs-block html-block sqs-block-html" data-block-type="2" id="block-yui_3_17_2_94_1509069313608_88531">
 								<div class="sqs-block-content">
-									<p> <strong>1.&nbsp;&nbsp;&nbsp;&nbsp; Construction of the plasmids</strong>
-									</p>
 									<p>
-										&nbsp; We constructed five plasmids-pEGFP-miR-21-sponge-6s; pEGFP-miR-21-sponge-2s; pHAGE-pre-miR-21; pSB1C3-miR-21-sponge-2s and pSB1C3-miR-21-sponge-6s. The plasmids of pEGFP-miR-21-sponge-6s and pEGFP-miR-21-sponge-2s were used to monitor the expression of miR-21 in cells. We also used pHAGE-pre-miR-21 to manipulate miR-21 expression in cells. And the plasmids of pSB1C3-miR-21-sponge-6s and pSB1C3-miR-21-sponge-2s were submitted to 2017IGEM (part number: BBa_K2514000 and BBa_K2514001).
+										<strong>
-									</p>
+											<!--[if gte vml 1]>
-									<p> <strong>1.1 Construction of pEGFP-miR-21-sponge-6s and pEGFP-miR-21-sponge-2s plasmid</strong>
+											<v:shape
-									</p>
+ id="_x0000_i1030" type="#_x0000_t75" style='width:198pt;height:107pt'>
-									<p>
+												<v:imagedata src="file:////Users/tianyuwang/Library/Group%20Containers/UBF8T346G9.Office/msoclip1/01/clip_image011.png"
-.1.1 Amplification of miR-21-sponge-6s and miR-21-sponge-2s fragment.
+&nbsp; o:title=""/>
+											</v:shape>
+											<![endif]-->
+										</strong>
+.2.2 &nbsp;Digested pHAGE-puro vector
 									</p>
 									<p>
-										To monitor the expression level of miR-21 in different cells, we made two plasmids of miR-21 sponges in the 3’UTR of GFP. Firstly, we designed miR-21 sponges contains six miR-21 binding sites with 3-nt spacers for bulged sites (pEGFP-miR-21-sponge-6s) and miR-21 sponges contains two miR-21 binding sites with 3-nt spacers for bulged sites (pEGFP-miR-21-sponge-2s) based on the sequence of hsa-miR-21 according to the previous study (Ebert MS, Neilson JR, Sharp PA. MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells. Nat Methods. 2007 Sep;4(9):721-6). Then we amplified miR-21-sponge-6s and miR-21-sponge-2s using primers. After that, we purified the PCR products by PCR Purification Kit and digested them with restriction enzymes Xho I and Hind III (Fig 1 A and B).
+										For
+										<a href="file:///D:/Users/Administrator/AppData/Local/Youdao/Dict/7.5.0.0/resultui/dict/?keyword=eukaryotic">eukaryotic</a>
+										<a href="file:///D:/Users/Administrator/AppData/Local/Youdao/Dict/7.5.0.0/resultui/dict/?keyword=expression">expression</a>
+										of has-miR-21 in cells, we digested the
+										<a href="file:///D:/Users/Administrator/AppData/Local/Youdao/Dict/7.5.0.0/resultui/dict/?keyword=eukaryotic">eukaryotic</a>
+										<a href="file:///D:/Users/Administrator/AppData/Local/Youdao/Dict/7.5.0.0/resultui/dict/?keyword=expression">expression</a>
+										vector pHAGE-puro with the same restriction enzymes Sal I and Not I&nbsp; (Fig 6).
 									</p>
 								</div>
 							</div>
-							<div class="sqs-block image-block sqs-block-image sqs-text-ready" data-block-type="5" id="block-yui_3_17_2_93_1509069313608_37436">
+							<div class="sqs-block image-block sqs-block-image sqs-text-ready" data-block-type="5" id="block-yui_3_17_2_94_1509069313608_105946">
-								<div class="sqs-block-content" id="yui_3_17_2_1_1509333291918_119">
+								<div class="sqs-block-content" id="yui_3_17_2_1_1509572305608_224">
-									<div class="image-block-outer-wrapper layout-caption-below design-layout-inline" id="yui_3_17_2_1_1509333291918_118">
+									<div class="image-block-outer-wrapper layout-caption-below design-layout-inline" id="yui_3_17_2_1_1509572305608_223">
-										<div class="intrinsic" style="max-width:468.0px;" id="yui_3_17_2_1_1509333291918_117">
+										<div class="intrinsic" style="max-width:198.0px;" id="yui_3_17_2_1_1509572305608_222">
-											<div style="padding-bottom: 29.0598%; overflow: hidden;" class="image-block-wrapper   has-aspect-ratio" data-description="<p> <em>Fig 1. Electrophoresis of miR-21-sponge-6s (A: lanes 1-3) and miR-21-sponge-2s (B: lanes 4-6) PCR product.</em>
+											<div style="padding-bottom: 54.5455%; overflow: hidden;" class="image-block-wrapper   has-aspect-ratio" data-description="<p>
+												<em>Fig 6. Electrophoresis of the digested pHAGE-puro vector.\</em>
 											</p>
-											" id="yui_3_17_2_1_1509333291918_116">
+											" id="yui_3_17_2_1_1509572305608_221">
 											<noscript>
-												<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ad39652deaab95256187/1509076299352/"  alt="Fig 1. Electrophoresis of miR-21-sponge-6s (A: lanes 1-3) and miR-21-sponge-2s (B: lanes 4-6) PCR product."  />
+												<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2af4f27ef2db556c92092/1509076855426/7.jpg"  alt="Fig 6. Electrophoresis of the digested pHAGE-puro vector.\"  />
 											</noscript>
-											<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ad39652deaab95256187/1509076299352/" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ad39652deaab95256187/1509076299352/" data-image-dimensions="468x136" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f2ad39652deaab95256187" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 1. Electrophoresis of miR-21-sponge-6s (A: lanes 1-3) and miR-21-sponge-2s (B: lanes 4-6) PCR product." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ad39652deaab95256187/1509076299352/?format=500w" data-image-resolution="500w"></div>
+											<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2af4f27ef2db556c92092/1509076855426/7.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2af4f27ef2db556c92092/1509076855426/7.jpg" data-image-dimensions="198x108" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f2af4f27ef2db556c92092" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 6. Electrophoresis of the digested pHAGE-puro vector.\" src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2af4f27ef2db556c92092/1509076855426/7.jpg?format=300w" data-image-resolution="300w"></div>
 										<div class="image-caption-wrapper">
 											<div class="image-caption">
-												<p> <em>Fig 1. Electrophoresis of miR-21-sponge-6s (A: lanes 1-3) and miR-21-sponge-2s (B: lanes 4-6) PCR product.</em>
+												<p>
+													<em>Fig 6. Electrophoresis of the digested pHAGE-puro vector.\</em>
 												</p>
 											</div>
@@ Line 286: / Line 609: @@
 							</div>
 						</div>
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 								</p>
-								<p>1.1.2 &nbsp;&nbsp;&nbsp; Digested pEGFP-C1 vector</p>
 								<p>
-									We know that microRNA can bind the 3 'UTR of genes to regulate the expression, so we choose EGFP to be the detector of miR-21. We digested the pEGFP-C1 vectors with restriction enzymes Xho I and Hind III (Fig 2).
+.2.3 Ligation of purified miR-21-precursor fragment to pHAGE-puro vector
+								</p>
+								<p>
+									To sequence the miR-21-precursor cloning we got, the purified miR-21-precursor fragment was ligated to pHAGE-puro vector. Then we selected the positive clones by PCR and sequencing (Fig 7 and 8).
 								</p>
 							</div>
 						</div>
-						<div class="sqs-block image-block sqs-block-image sqs-text-ready" data-block-type="5" id="block-yui_3_17_2_93_1509069313608_68774">
+						<div class="sqs-block image-block sqs-block-image sqs-text-ready" data-block-type="5" id="block-yui_3_17_2_94_1509069313608_121180">
-							<div class="sqs-block-content" id="yui_3_17_2_1_1509333291918_139">
+							<div class="sqs-block-content" id="yui_3_17_2_1_1509572305608_241">
-								<div class="image-block-outer-wrapper layout-caption-below design-layout-inline" id="yui_3_17_2_1_1509333291918_138">
+								<div class="image-block-outer-wrapper layout-caption-below design-layout-inline" id="yui_3_17_2_1_1509572305608_240">
-									<div class="intrinsic" style="max-width:193.0px;" id="yui_3_17_2_1_1509333291918_137">
+									<div class="intrinsic" style="max-width:192.0px;" id="yui_3_17_2_1_1509572305608_239">
-										<div style="padding-bottom: 49.7409%; overflow: hidden;" class="image-block-wrapper   has-aspect-ratio" data-description="<p>
+										<div style="padding-bottom: 55.7292%; overflow: hidden;" class="image-block-wrapper   has-aspect-ratio" data-description="<p>A</p>
-											<em>Fig 2. Electrophoresis of the digested pEGFP-c1 vector.</em>
+										" id="yui_3_17_2_1_1509572305608_238">
-										</p>
-										" id="yui_3_17_2_1_1509333291918_136">
 										<noscript>
-											<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ad766c3194b973e71691/1509076361545/Fig+2.+Electrophoresis+of+the+digested+pEGFP-c1+vector."  alt="Fig 2. Electrophoresis of the digested pEGFP-c1 vector."  />
+											<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2aff29140b7ad7ea383e0/1509076986534/8.jpg"  alt="A"  />
 										</noscript>
-										<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ad766c3194b973e71691/1509076361545/Fig+2.+Electrophoresis+of+the+digested+pEGFP-c1+vector." data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ad766c3194b973e71691/1509076361545/Fig+2.+Electrophoresis+of+the+digested+pEGFP-c1+vector." data-image-dimensions="193x96" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f2ad766c3194b973e71691" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 2. Electrophoresis of the digested pEGFP-c1 vector." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ad766c3194b973e71691/1509076361545/Fig+2.+Electrophoresis+of+the+digested+pEGFP-c1+vector.?format=300w" data-image-resolution="300w"></div>
+										<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2aff29140b7ad7ea383e0/1509076986534/8.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2aff29140b7ad7ea383e0/1509076986534/8.jpg" data-image-dimensions="192x107" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f2aff29140b7ad7ea383e0" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="A" src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2aff29140b7ad7ea383e0/1509076986534/8.jpg?format=300w" data-image-resolution="300w"></div>
 									<div class="image-caption-wrapper">
 										<div class="image-caption">
-											<p>
+											<p>A</p>
-												<em>Fig 2. Electrophoresis of the digested pEGFP-c1 vector.</em>
-											</p>
 										</div>
 									</div>
@@ Line 366: / Line 654: @@
 						</div>
 					</div>
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-								<![endif]-->
-							</p>
-							<p>
-.1.3 &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Ligation of purified miR-21-sponge-6s and miR-21-sponge-2s fragments&nbsp; to pEGFP-c1 vector
-							</p>
-							<p>
-								miR-21-sponge-6s fragments and miR-21-sponge-2s fragments were ligated to pEGFP-c1 vector, respectively. Then we selected the positive clones by PCR and sequencing (Fig 3 and 4)
-							</p>
-						</div>
-					</div>
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-							<div class="image-block-outer-wrapper layout-caption-below design-layout-inline" id="yui_3_17_2_1_1509333291918_172">
+							<div class="image-block-outer-wrapper layout-caption-below design-layout-inline" id="yui_3_17_2_1_1509572305608_257">
-								<div class="intrinsic" style="max-width:318.0px;" id="yui_3_17_2_1_1509333291918_171">
+								<div class="intrinsic" style="max-width:340.0px;" id="yui_3_17_2_1_1509572305608_256">
-									<div style="padding-bottom: 55.9748%; overflow: hidden;" class="image-block-wrapper   has-aspect-ratio" data-description="<p>C</p>
+									<div style="padding-bottom: 20.8824%; overflow: hidden;" class="image-block-wrapper   has-aspect-ratio" data-description="<p>B</p>
 									<p>
 										<em>
-											Fig 3. Electrophoresis of the PCR identifications of pEGFP- miR-21-sponge-6s (A: lane 1-3) and&amp;nbsp; pEGFP- miR-21-sponge-2s (B: lane 4-6) vector and sequencing map.
+											Fig 7. Electrophoresis of the PCR identified production of pHAGE- pre-miR-21 vector (A) and sequencing map (B) .
 										</em>
 									</p>
-									" id="yui_3_17_2_1_1509333291918_170">
+									<p>&nbsp;</p>
+									" id="yui_3_17_2_1_1509572305608_255">
 									<noscript>
-										<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ae499140b7ad7ea3604f/1509076642038/"  alt="C Fig 3. Electrophoresis of the PCR identifications of pEGFP- miR-21-sponge-6s (A: lane 1-3) and  pEGFP- miR-21-sponge-2s (B: lane 4-6) vector and sequencing map."  />
+										<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2b02ae31d19b2c6be28b0/1509077035734/"  alt="B Fig 7. Electrophoresis of the PCR identified production of pHAGE- pre-miR-21 vector (A) and sequencing map (B) .  "  />
 									</noscript>
-									<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ae499140b7ad7ea3604f/1509076642038/" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ae499140b7ad7ea3604f/1509076642038/" data-image-dimensions="318x178" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f2ae499140b7ad7ea3604f" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="C Fig 3. Electrophoresis of the PCR identifications of pEGFP- miR-21-sponge-6s (A: lane 1-3) and&nbsp; pEGFP- miR-21-sponge-2s (B: lane 4-6) vector and sequencing map." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ae499140b7ad7ea3604f/1509076642038/?format=500w" data-image-resolution="500w"></div>
+									<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2b02ae31d19b2c6be28b0/1509077035734/" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2b02ae31d19b2c6be28b0/1509077035734/" data-image-dimensions="340x71" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f2b02ae31d19b2c6be28b0" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="B Fig 7. Electrophoresis of the PCR identified production of pHAGE- pre-miR-21 vector (A) and sequencing map (B) . &nbsp;" src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2b02ae31d19b2c6be28b0/1509077035734/?format=500w" data-image-resolution="500w"></div>
 								<div class="image-caption-wrapper">
 									<div class="image-caption">
-										<p>C</p>
+										<p>B</p>
 										<p>
 											<em>
-												Fig 3. Electrophoresis of the PCR identifications of pEGFP- miR-21-sponge-6s (A: lane 1-3) and&nbsp; pEGFP- miR-21-sponge-2s (B: lane 4-6) vector and sequencing map.
+												Fig 7. Electrophoresis of the PCR identified production of pHAGE- pre-miR-21 vector (A) and sequencing map (B) .
 											</em>
 										</p>
+										<p>&nbsp;</p>
 									</div>
 								</div>
@@ Line 440: / Line 692: @@
 					</div>
 				</div>
-				<div class="sqs-block image-block sqs-block-image sqs-text-ready" data-block-type="5" id="block-yui_3_17_2_94_1509069313608_21117">
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+					<div class="sqs-block-content" id="yui_3_17_2_1_1509572305608_275">
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+						<div class="image-block-outer-wrapper layout-caption-below design-layout-inline" id="yui_3_17_2_1_1509572305608_274">
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+							<div class="intrinsic" style="max-width:182.0px;" id="yui_3_17_2_1_1509572305608_273">
-								<div style="padding-bottom: 55.9585%; overflow: hidden;" class="image-block-wrapper   has-aspect-ratio" data-description="<p>
+								<div style="padding-bottom: 54.3956%; overflow: hidden;" class="image-block-wrapper   has-aspect-ratio" data-description="<p>
-									<em>
+									<em>Fig 8. Electrophoresis of pHAGE-pre-miR-21 vector.</em>
-										Fig 4. Electrophoresis of pEGFP- miR-21-sponge-6s (lane 1,2) and&amp;nbsp; pEGFP- miR-21-sponge-2s (lane 3,4) vector.
-									</em>
 								</p>
-								" id="yui_3_17_2_1_1509333291918_187">
+								" id="yui_3_17_2_1_1509572305608_272">
 								<noscript>
-									<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ae59652deaab95257722/1509076660513/"  alt="Fig 4. Electrophoresis of pEGFP- miR-21-sponge-6s (lane 1,2) and  pEGFP- miR-21-sponge-2s (lane 3,4) vector."  />
+									<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2b0809140b7ad7ea3904a/1509077122116/"  alt="Fig 8. Electrophoresis of pHAGE-pre-miR-21 vector."  />
 								</noscript>
-								<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ae59652deaab95257722/1509076660513/" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ae59652deaab95257722/1509076660513/" data-image-dimensions="193x108" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f2ae59652deaab95257722" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 4. Electrophoresis of pEGFP- miR-21-sponge-6s (lane 1,2) and&nbsp; pEGFP- miR-21-sponge-2s (lane 3,4) vector." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2ae59652deaab95257722/1509076660513/?format=300w" data-image-resolution="300w"></div>
+								<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2b0809140b7ad7ea3904a/1509077122116/" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2b0809140b7ad7ea3904a/1509077122116/" data-image-dimensions="182x99" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f2b0809140b7ad7ea3904a" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 8. Electrophoresis of pHAGE-pre-miR-21 vector." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2b0809140b7ad7ea3904a/1509077122116/?format=300w" data-image-resolution="300w"></div>
 							<div class="image-caption-wrapper">
 								<div class="image-caption">
 									<p>
-										<em>
+										<em>Fig 8. Electrophoresis of pHAGE-pre-miR-21 vector.</em>
-											Fig 4. Electrophoresis of pEGFP- miR-21-sponge-6s (lane 1,2) and&nbsp; pEGFP- miR-21-sponge-2s (lane 3,4) vector.
-										</em>
 									</p>
 								</div>
@@ Line 474: / Line 722: @@
 				</div>
 			</div>
-			<div class="sqs-block html-block sqs-block-html" data-block-type="2" id="block-yui_3_17_2_94_1509069313608_12051">
+			<div class="sqs-block html-block sqs-block-html" data-block-type="2" id="block-yui_3_17_2_94_1509069313608_178776">
 				<div class="sqs-block-content">
 					<p>
-						<!--[if gte vml 1]>
+						<strong>
-						<v:shape
+							<!--[if gte vml 1]>
-  id="_x0000_i1027" type="#_x0000_t75" style='width:281pt;height:214pt'>
+							<v:shape id="图片_x0020_1"
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+  o:spid="_x0000_i1033" type="#_x0000_t75" style='width:337pt;height:71pt;
+ visibility:visible'>
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 &nbsp; o:title=""/>
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+							</v:shape>
-						<![endif]-->
+							<![endif]-->
+.3 &nbsp;Construction of pSB-miR-21-sponge-6s and pSB-miR-21-sponge-2s plasmid
+						</strong>
+						<br>
+.3.1 Amplification of miR-21-sponge-6s and miR-21-sponge-2s fragment.
 					</p>
 					<p>
 						<!--[if gte vml 1]>
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+ height:98pt;visibility:visible'>
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 &nbsp; o:title=""/>
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@@ Line 495: / Line 750: @@
 					</p>
 					<p>
-						<strong>1.2 &nbsp;&nbsp;&nbsp; Construction of miR-21- precuser expression plasmid</strong>
+						In order to submit the parts to IGM2017, we amplified miR-21-sponge-6s and miR-21-sponge-2s using primers. After that, we purified the PCR products by PCR Purification Kit and digested them with restriction enzymes EcoR I and Pst I (Fig 9 A and B).
-					</p>
-					<p>
-.2.1 &nbsp;&nbsp;&nbsp;&nbsp; Amplification of miR-21- precuser fragment.
-					</p>
-					<p>
-						<strong>
-							<!--[if gte vml 1]>
-							<v:shape
- id="_x0000_i1029" type="#_x0000_t75" style='width:193pt;height:108pt'>
-								<v:imagedata src="file:////Users/tianyuwang/Library/Group%20Containers/UBF8T346G9.Office/msoclip1/01/clip_image009.png"
-&nbsp; o:title=""/>
-							</v:shape>
-							<![endif]-->
-						</strong>
-						To set up a model to determent the amount of miR-21 in cells, we made a construct to express miR-21.&nbsp; We designed the specific primers of human miR-21-precuser based on the sequences of miR-21 precuser (
-						<a href="http://www.mirbase.org/cgi-bin/mirna_entry.pl?acc=MI0000077">http://www.miRbase.org/cgi-bin/miRna_entry.pl?acc=MI0000077</a>
-						) and amplified 72bp fragment by PCR. After that, we digested the purified PCR products with restriction enzymes Sal I and Not I (Fig 5).
 					</p>
 				</div>
 			</div>
-			<div class="sqs-block image-block sqs-block-image sqs-text-ready" data-block-type="5" id="block-yui_3_17_2_94_1509069313608_88253">
+			<div class="sqs-block image-block sqs-block-image sqs-text-ready" data-block-type="5" id="block-yui_3_17_2_4_1509162483656_19010">
-				<div class="sqs-block-content" id="yui_3_17_2_1_1509333291918_207">
+				<div class="sqs-block-content" id="yui_3_17_2_1_1509572305608_292">
-					<div class="image-block-outer-wrapper layout-caption-below design-layout-inline" id="yui_3_17_2_1_1509333291918_206">
+					<div class="image-block-outer-wrapper layout-caption-below design-layout-inline" id="yui_3_17_2_1_1509572305608_291">
-						<div class="intrinsic" style="max-width:198.0px;" id="yui_3_17_2_1_1509333291918_205">
+						<div class="intrinsic" style="max-width:564.0px;" id="yui_3_17_2_1_1509572305608_290">
-							<div style="padding-bottom: 54.0404%; overflow: hidden;" class="image-block-wrapper   has-aspect-ratio" data-description="<p>
+							<div style="padding-bottom: 28.0142%; overflow: hidden;" class="image-block-wrapper   has-aspect-ratio" data-description="<p>
 								<em>
-									Fig 5. Electrophoresis of miR-21-precouser PCR product. The lanes 1-3 showed the miR-21-precouser bands.
+									Fig 9. Electrophoresis of miR-21-sponge-6s (A: lanes 1-3) and miR-21-sponge-2s (B: lanes 4-6) PCR product.
 								</em>
 							</p>
-							" id="yui_3_17_2_1_1509333291918_204">
+							" id="yui_3_17_2_1_1509572305608_289">
 							<noscript>
-								<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2af200d9297adf936234e/1509076769342/6.jpg"  alt="Fig 5. Electrophoresis of miR-21-precouser PCR product. The lanes 1-3 showed the miR-21-precouser bands."  />
+								<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f4028a652dea26877dff2d/1509163665493/9a.jpg"  alt="Fig 9. Electrophoresis of miR-21-sponge-6s (A: lanes 1-3) and miR-21-sponge-2s (B: lanes 4-6) PCR product."  />
 							</noscript>
-							<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2af200d9297adf936234e/1509076769342/6.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2af200d9297adf936234e/1509076769342/6.jpg" data-image-dimensions="198x107" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f2af200d9297adf936234e" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 5. Electrophoresis of miR-21-precouser PCR product. The lanes 1-3 showed the miR-21-precouser bands." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2af200d9297adf936234e/1509076769342/6.jpg?format=300w" data-image-resolution="300w"></div>
+							<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f4028a652dea26877dff2d/1509163665493/9a.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f4028a652dea26877dff2d/1509163665493/9a.jpg" data-image-dimensions="564x158" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f4028a652dea26877dff2d" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 9. Electrophoresis of miR-21-sponge-6s (A: lanes 1-3) and miR-21-sponge-2s (B: lanes 4-6) PCR product." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f4028a652dea26877dff2d/1509163665493/9a.jpg?format=750w" data-image-resolution="750w"></div>
 						<div class="image-caption-wrapper">
@@ Line 538: / Line 776: @@
 								<p>
 									<em>
-										Fig 5. Electrophoresis of miR-21-precouser PCR product. The lanes 1-3 showed the miR-21-precouser bands.
+										Fig 9. Electrophoresis of miR-21-sponge-6s (A: lanes 1-3) and miR-21-sponge-2s (B: lanes 4-6) PCR product.
 									</em>
 								</p>
@@ Line 550: / Line 788: @@
 			</div>
 		</div>
-		<div class="sqs-block html-block sqs-block-html" data-block-type="2" id="block-yui_3_17_2_94_1509069313608_88531">
+		<div class="sqs-block html-block sqs-block-html" data-block-type="2" id="block-yui_3_17_2_4_1509162483656_19289">
 			<div class="sqs-block-content">
+				<p>1.3.2 &nbsp;&nbsp;&nbsp;&nbsp; Digested pSB1C3 vector</p>
 				<p>
-					<strong>
+					<!--[if gte vml 1]>
-						<!--[if gte vml 1]>
+					<v:shape id="_x0000_i1035" type="#_x0000_t75"
-						<v:shape
+ style='width:415pt;height:116pt'>
- id="_x0000_i1030" type="#_x0000_t75" style='width:198pt;height:107pt'>
+						<v:imagedata src="file:////Users/tianyuwang/Library/Group%20Containers/UBF8T346G9.Office/msoclip1/01/clip_image021.png"
-							<v:imagedata src="file:////Users/tianyuwang/Library/Group%20Containers/UBF8T346G9.Office/msoclip1/01/clip_image011.png"
 &nbsp; o:title=""/>
-						</v:shape>
+					</v:shape>
-						<![endif]-->
+					<![endif]-->
-					</strong>
-.2.2 &nbsp;Digested pHAGE-puro vector
 				</p>
 				<p>
-					For
+					We digested the pSB1C3 vector with the same restriction enzymes EcoR I and Pst I&nbsp; (Fig 10).
-					<a href="file:///D:/Users/Administrator/AppData/Local/Youdao/Dict/7.5.0.0/resultui/dict/?keyword=eukaryotic">eukaryotic</a>
-					<a href="file:///D:/Users/Administrator/AppData/Local/Youdao/Dict/7.5.0.0/resultui/dict/?keyword=expression">expression</a>
-					of has-miR-21 in cells, we digested the
-					<a href="file:///D:/Users/Administrator/AppData/Local/Youdao/Dict/7.5.0.0/resultui/dict/?keyword=eukaryotic">eukaryotic</a>
-					<a href="file:///D:/Users/Administrator/AppData/Local/Youdao/Dict/7.5.0.0/resultui/dict/?keyword=expression">expression</a>
-					vector pHAGE-puro with the same restriction enzymes Sal I and Not I&nbsp; (Fig 6).
 				</p>
 			</div>
 		</div>
-		<div class="sqs-block image-block sqs-block-image sqs-text-ready" data-block-type="5" id="block-yui_3_17_2_94_1509069313608_105946">
+		<div class="sqs-block image-block sqs-block-image sqs-text-ready" data-block-type="5" id="block-yui_3_17_2_4_1509162483656_30874">
-			<div class="sqs-block-content" id="yui_3_17_2_1_1509333291918_224">
+			<div class="sqs-block-content" id="yui_3_17_2_1_1509572305608_309">
-				<div class="image-block-outer-wrapper layout-caption-below design-layout-inline" id="yui_3_17_2_1_1509333291918_223">
+				<div class="image-block-outer-wrapper layout-caption-below design-layout-inline" id="yui_3_17_2_1_1509572305608_308">
-					<div class="intrinsic" style="max-width:198.0px;" id="yui_3_17_2_1_1509333291918_222">
+					<div class="intrinsic" style="max-width:268.0px;" id="yui_3_17_2_1_1509572305608_307">
-						<div style="padding-bottom: 54.5455%; overflow: hidden;" class="image-block-wrapper   has-aspect-ratio" data-description="<p>
+						<div style="padding-bottom: 56.7164%; overflow: hidden;" class="image-block-wrapper   has-aspect-ratio" data-description="<p>
-							<em>Fig 6. Electrophoresis of the digested pHAGE-puro vector.\</em>
+							<em>Fig 10. Electrophoresis of the digested pSB1C3 vector.</em>
 						</p>
-						" id="yui_3_17_2_1_1509333291918_221">
+						" id="yui_3_17_2_1_1509572305608_306">
 						<noscript>
-							<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2af4f27ef2db556c92092/1509076855426/7.jpg"  alt="Fig 6. Electrophoresis of the digested pHAGE-puro vector.\"  />
+							<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f402b36926700aba0d0796/1509163705061/10.jpg"  alt="Fig 10. Electrophoresis of the digested pSB1C3 vector."  />
 						</noscript>
-						<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2af4f27ef2db556c92092/1509076855426/7.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2af4f27ef2db556c92092/1509076855426/7.jpg" data-image-dimensions="198x108" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f2af4f27ef2db556c92092" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 6. Electrophoresis of the digested pHAGE-puro vector.\" src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2af4f27ef2db556c92092/1509076855426/7.jpg?format=300w" data-image-resolution="300w"></div>
+						<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f402b36926700aba0d0796/1509163705061/10.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f402b36926700aba0d0796/1509163705061/10.jpg" data-image-dimensions="268x152" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f402b36926700aba0d0796" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 10. Electrophoresis of the digested pSB1C3 vector." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f402b36926700aba0d0796/1509163705061/10.jpg?format=300w" data-image-resolution="300w"></div>
 					<div class="image-caption-wrapper">
 						<div class="image-caption">
 							<p>
-								<em>Fig 6. Electrophoresis of the digested pHAGE-puro vector.\</em>
+								<em>Fig 10. Electrophoresis of the digested pSB1C3 vector.</em>
 							</p>
 						</div>
@@ Line 605: / Line 835: @@
 		</div>
 	</div>
-	<div class="sqs-block html-block sqs-block-html" data-block-type="2" id="block-yui_3_17_2_94_1509069313608_106224">
+	<div class="sqs-block html-block sqs-block-html" data-block-type="2" id="block-yui_3_17_2_4_1509162483656_31152">
 		<div class="sqs-block-content">
 			<p>
 				<!--[if gte vml 1]>
 				<v:shape
-  id="_x0000_i1031" type="#_x0000_t75" style='width:198pt;height:107pt'>
+  id="_x0000_i1036" type="#_x0000_t75" style='width:201pt;height:114pt'>
-					<v:imagedata src="file:////Users/tianyuwang/Library/Group%20Containers/UBF8T346G9.Office/msoclip1/01/clip_image013.png"
+					<v:imagedata src="file:////Users/tianyuwang/Library/Group%20Containers/UBF8T346G9.Office/msoclip1/01/clip_image023.png"
 &nbsp; o:title=""/>
 				</v:shape>
@@ Line 617: / Line 847: @@
 			</p>
 			<p>
-.2.3 Ligation of purified miR-21-precuser fragment to pHAGE-puro vector
+.3.3 &nbsp;&nbsp;&nbsp;&nbsp; Ligation of purified miR-21-sponge-6s and miR-21-sponge-2s fragment&nbsp; to pSB1C3 vector
 			</p>
 			<p>
-				To sequence the miR-21-precuser cloning we got, the purified miR-21-precuser fragment was ligated to pHAGE-puro vector. Then we selected the positive clones by PCR and sequencing (Fig 7 and 8).
+				We ligated the purified miR-21-sponge-6s or miR-21-sponge-2s fragment to pSB1C3 vector, respectively. Then we selected the positive clones by PCR and sequencing (Fig 11 and 12).
 			</p>
 		</div>
 	</div>
-	<div class="sqs-block image-block sqs-block-image sqs-text-ready" data-block-type="5" id="block-yui_3_17_2_94_1509069313608_121180">
+	<div class="sqs-block image-block sqs-block-image sqs-text-ready" data-block-type="5" id="block-yui_3_17_2_4_1509162483656_49246">
-		<div class="sqs-block-content" id="yui_3_17_2_1_1509333291918_241">
+		<div class="sqs-block-content" id="yui_3_17_2_1_1509572305608_326">
-			<div class="image-block-outer-wrapper layout-caption-below design-layout-inline" id="yui_3_17_2_1_1509333291918_240">
+			<div class="image-block-outer-wrapper layout-caption-below design-layout-inline" id="yui_3_17_2_1_1509572305608_325">
-				<div class="intrinsic" style="max-width:192.0px;" id="yui_3_17_2_1_1509333291918_239">
+				<div class="intrinsic" style="max-width:560.0px;" id="yui_3_17_2_1_1509572305608_324">
-					<div style="padding-bottom: 55.7292%; overflow: hidden;" class="image-block-wrapper   has-aspect-ratio" data-description="<p>A</p>
+					<div style="padding-bottom: 29.4643%; overflow: hidden;" class="image-block-wrapper   has-aspect-ratio" data-description="" id="yui_3_17_2_1_1509572305608_323">
-					" id="yui_3_17_2_1_1509333291918_238">
+						<noscript>
-					<noscript>
+							<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f402f510952624f5425864/1509163778597/11ab.jpg"  alt="11ab.jpg"  />
-						<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2aff29140b7ad7ea383e0/1509076986534/8.jpg"  alt="A"  />
+						</noscript>
-					</noscript>
+						<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f402f510952624f5425864/1509163778597/11ab.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f402f510952624f5425864/1509163778597/11ab.jpg" data-image-dimensions="560x165" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f402f510952624f5425864" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="11ab.jpg" src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f402f510952624f5425864/1509163778597/11ab.jpg?format=750w" data-image-resolution="750w"></div>
-					<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2aff29140b7ad7ea383e0/1509076986534/8.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2aff29140b7ad7ea383e0/1509076986534/8.jpg" data-image-dimensions="192x107" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f2aff29140b7ad7ea383e0" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="A" src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2aff29140b7ad7ea383e0/1509076986534/8.jpg?format=300w" data-image-resolution="300w"></div>
-				<div class="image-caption-wrapper">
-					<div class="image-caption">
-						<p>A</p>
-					</div>
 				</div>
@@ Line 647: / Line 872: @@
 		</div>
 	</div>
-</div>
+	<div class="sqs-block image-block sqs-block-image sqs-text-ready" data-block-type="5" id="block-yui_3_17_2_4_1509162483656_52024">
-<div class="sqs-block image-block sqs-block-image sqs-text-ready" data-block-type="5" id="block-yui_3_17_2_94_1509069313608_139550">
+		<div class="sqs-block-content" id="yui_3_17_2_1_1509572305608_343">
-	<div class="sqs-block-content" id="yui_3_17_2_1_1509333291918_258">
-		<div class="image-block-outer-wrapper layout-caption-below design-layout-inline" id="yui_3_17_2_1_1509333291918_257">
+			<div class="image-block-outer-wrapper layout-caption-below design-layout-inline" id="yui_3_17_2_1_1509572305608_342">
-			<div class="intrinsic" style="max-width:340.0px;" id="yui_3_17_2_1_1509333291918_256">
+				<div class="intrinsic" style="max-width:696.0px;" id="yui_3_17_2_1_1509572305608_341">
-				<div style="padding-bottom: 20.8824%; overflow: hidden;" class="image-block-wrapper   has-aspect-ratio" data-description="<p>B</p>
+					<div style="padding-bottom: 38.0747%; overflow: hidden;" class="image-block-wrapper   has-aspect-ratio" data-description="" id="yui_3_17_2_1_1509572305608_340">
-				<p>
+						<noscript>
-					<em>
+							<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403102774d1138b78c19a/1509163808883/11c.jpg"  alt="11c.jpg"  />
-						Fig 7. Electrophoresis of the PCR identified production of pHAGE- pre-miR-21 vector (A) and sequencing map (B) .
+						</noscript>
-					</em>
+						<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403102774d1138b78c19a/1509163808883/11c.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403102774d1138b78c19a/1509163808883/11c.jpg" data-image-dimensions="696x265" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f403102774d1138b78c19a" data-type="image" data-position-mode="standard" style="left: 0%; top: -0.0781863%; width: 100%; height: 100.156%; position: absolute;" alt="11c.jpg" src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403102774d1138b78c19a/1509163808883/11c.jpg?format=750w" data-image-resolution="750w"></div>
-				</p>
-				<p>&nbsp;</p>
-				" id="yui_3_17_2_1_1509333291918_255">
-				<noscript>
-					<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2b02ae31d19b2c6be28b0/1509077035734/"  alt="B Fig 7. Electrophoresis of the PCR identified production of pHAGE- pre-miR-21 vector (A) and sequencing map (B) .  "  />
-				</noscript>
-				<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2b02ae31d19b2c6be28b0/1509077035734/" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2b02ae31d19b2c6be28b0/1509077035734/" data-image-dimensions="340x71" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f2b02ae31d19b2c6be28b0" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="B Fig 7. Electrophoresis of the PCR identified production of pHAGE- pre-miR-21 vector (A) and sequencing map (B) . &nbsp;" src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2b02ae31d19b2c6be28b0/1509077035734/?format=500w" data-image-resolution="500w"></div>
-			<div class="image-caption-wrapper">
-				<div class="image-caption">
-					<p>B</p>
-					<p>
-						<em>
-							Fig 7. Electrophoresis of the PCR identified production of pHAGE- pre-miR-21 vector (A) and sequencing map (B) .
-						</em>
-					</p>
-					<p>&nbsp;</p>
 				</div>
 			</div>
 		</div>
 	</div>
+	<div class="sqs-block image-block sqs-block-image sqs-text-ready" data-block-type="5" id="block-yui_3_17_2_4_1509162483656_59772">
+		<div class="sqs-block-content" id="yui_3_17_2_1_1509572305608_360">
-</div>
+			<div class="image-block-outer-wrapper layout-caption-below design-layout-inline" id="yui_3_17_2_1_1509572305608_359">
-</div>
-<div class="sqs-block image-block sqs-block-image sqs-text-ready" data-block-type="5" id="block-yui_3_17_2_94_1509069313608_178499">
-<div class="sqs-block-content" id="yui_3_17_2_1_1509333291918_275">
-	<div class="image-block-outer-wrapper layout-caption-below design-layout-inline" id="yui_3_17_2_1_1509333291918_274">
+				<div class="intrinsic" style="max-width:259.0px;" id="yui_3_17_2_1_1509572305608_358">
-		<div class="intrinsic" style="max-width:182.0px;" id="yui_3_17_2_1_1509333291918_273">
+					<div style="padding-bottom: 59.4595%; overflow: hidden;" class="image-block-wrapper   has-aspect-ratio" data-description="<p>
+						<em>
+							Fig 12. Electrophoresis of pSB1C3- miR-21-sponge-6s (lane 1, 2) and&amp;nbsp; pSB1C3- miR-21-sponge-2s (lane 3, 4) vector.
+						</em>
+					</p>
+					" id="yui_3_17_2_1_1509572305608_357">
+					<noscript>
+						<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403336c31940534284984/1509163831033/12.jpg"  alt="Fig 12. Electrophoresis of pSB1C3- miR-21-sponge-6s (lane 1, 2) and  pSB1C3- miR-21-sponge-2s (lane 3, 4) vector."  />
+					</noscript>
+					<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403336c31940534284984/1509163831033/12.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403336c31940534284984/1509163831033/12.jpg" data-image-dimensions="259x154" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f403336c31940534284984" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 12. Electrophoresis of pSB1C3- miR-21-sponge-6s (lane 1, 2) and&nbsp; pSB1C3- miR-21-sponge-2s (lane 3, 4) vector." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403336c31940534284984/1509163831033/12.jpg?format=300w" data-image-resolution="300w"></div>
-			<div style="padding-bottom: 54.3956%; overflow: hidden;" class="image-block-wrapper   has-aspect-ratio" data-description="<p>
+				<div class="image-caption-wrapper">
-				<em>Fig 8. Electrophoresis of pHAGE-pre-miR-21 vector.</em>
+					<div class="image-caption">
-			</p>
+						<p>
-			" id="yui_3_17_2_1_1509333291918_272">
+							<em>
-			<noscript>
+								Fig 12. Electrophoresis of pSB1C3- miR-21-sponge-6s (lane 1, 2) and&nbsp; pSB1C3- miR-21-sponge-2s (lane 3, 4) vector.
-				<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2b0809140b7ad7ea3904a/1509077122116/"  alt="Fig 8. Electrophoresis of pHAGE-pre-miR-21 vector."  />
+							</em>
-			</noscript>
+						</p>
-			<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2b0809140b7ad7ea3904a/1509077122116/" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2b0809140b7ad7ea3904a/1509077122116/" data-image-dimensions="182x99" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f2b0809140b7ad7ea3904a" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 8. Electrophoresis of pHAGE-pre-miR-21 vector." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f2b0809140b7ad7ea3904a/1509077122116/?format=300w" data-image-resolution="300w"></div>
+					</div>
+				</div>
-		<div class="image-caption-wrapper">
-			<div class="image-caption">
-				<p>
-					<em>Fig 8. Electrophoresis of pHAGE-pre-miR-21 vector.</em>
-				</p>
 			</div>
-		</div>
-	</div>
-</div>
-</div>
-</div>
-<div class="sqs-block html-block sqs-block-html" data-block-type="2" id="block-yui_3_17_2_94_1509069313608_178776">
-<div class="sqs-block-content">
-<p>
-	<strong>
-		<!--[if gte vml 1]>
-		<v:shape id="图片_x0020_1"
- o:spid="_x0000_i1033" type="#_x0000_t75" style='width:337pt;height:71pt;
- visibility:visible'>
-			<v:imagedata src="file:////Users/tianyuwang/Library/Group%20Containers/UBF8T346G9.Office/msoclip1/01/clip_image017.png"
-&nbsp; o:title=""/>
-		</v:shape>
-		<![endif]-->
-.3 &nbsp;Construction of pSB-miR-21-sponge-6s and pSB-miR-21-sponge-2s plasmid
-	</strong>
-	<br>
-.3.1 Amplification of miR-21-sponge-6s and miR-21-sponge-2s fragment.
-</p>
-<p>
-	<!--[if gte vml 1]>
-	<v:shape
- id="图片_x0020_2" o:spid="_x0000_i1034" type="#_x0000_t75" style='width:181pt;
- height:98pt;visibility:visible'>
-		<v:imagedata src="file:////Users/tianyuwang/Library/Group%20Containers/UBF8T346G9.Office/msoclip1/01/clip_image019.png"
-&nbsp; o:title=""/>
-	</v:shape>
-	<![endif]-->
-</p>
-<p>
-	In order to submit the parts to IGM2017, we amplified miR-21-sponge-6s and miR-21-sponge-2s using primers. After that, we purified the PCR products by PCR Purification Kit and digested them with restriction enzymes EcoR I and Pst I (Fig 9 A and B).
-</p>
-</div>
-</div>
-<div class="sqs-block image-block sqs-block-image sqs-text-ready" data-block-type="5" id="block-yui_3_17_2_4_1509162483656_19010">
-<div class="sqs-block-content" id="yui_3_17_2_1_1509333291918_292">
-<div class="image-block-outer-wrapper layout-caption-below design-layout-inline" id="yui_3_17_2_1_1509333291918_291">
-	<div class="intrinsic" style="max-width:564.0px;" id="yui_3_17_2_1_1509333291918_290">
-		<div style="padding-bottom: 28.0142%; overflow: hidden;" class="image-block-wrapper   has-aspect-ratio" data-description="<p>
-			<em>
-				Fig 9. Electrophoresis of miR-21-sponge-6s (A: lanes 1-3) and miR-21-sponge-2s (B: lanes 4-6) PCR product.
-			</em>
-		</p>
-		" id="yui_3_17_2_1_1509333291918_289">
-		<noscript>
-			<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f4028a652dea26877dff2d/1509163665493/9a.jpg"  alt="Fig 9. Electrophoresis of miR-21-sponge-6s (A: lanes 1-3) and miR-21-sponge-2s (B: lanes 4-6) PCR product."  />
-		</noscript>
-		<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f4028a652dea26877dff2d/1509163665493/9a.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f4028a652dea26877dff2d/1509163665493/9a.jpg" data-image-dimensions="564x158" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f4028a652dea26877dff2d" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 9. Electrophoresis of miR-21-sponge-6s (A: lanes 1-3) and miR-21-sponge-2s (B: lanes 4-6) PCR product." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f4028a652dea26877dff2d/1509163665493/9a.jpg?format=750w" data-image-resolution="750w"></div>
-	<div class="image-caption-wrapper">
-		<div class="image-caption">
-			<p>
-				<em>
-					Fig 9. Electrophoresis of miR-21-sponge-6s (A: lanes 1-3) and miR-21-sponge-2s (B: lanes 4-6) PCR product.
-				</em>
-			</p>
 		</div>
-	</div>
-</div>
-</div>
-</div>
-</div>
-<div class="sqs-block html-block sqs-block-html" data-block-type="2" id="block-yui_3_17_2_4_1509162483656_19289">
-<div class="sqs-block-content">
-<p>1.3.2 &nbsp;&nbsp;&nbsp;&nbsp; Digested pSB1C3 vector</p>
-<p>
-<!--[if gte vml 1]>
-<v:shape id="_x0000_i1035" type="#_x0000_t75"
- style='width:415pt;height:116pt'>
-	<v:imagedata src="file:////Users/tianyuwang/Library/Group%20Containers/UBF8T346G9.Office/msoclip1/01/clip_image021.png"
-&nbsp; o:title=""/>
-</v:shape>
-<![endif]-->
-</p>
-<p>
-We digested the pSB1C3 vector with the same restriction enzymes EcoR I and Pst I&nbsp; (Fig 10).
-</p>
-</div>
-</div>
-<div class="sqs-block image-block sqs-block-image sqs-text-ready" data-block-type="5" id="block-yui_3_17_2_4_1509162483656_30874">
-<div class="sqs-block-content" id="yui_3_17_2_1_1509333291918_309">
-<div class="image-block-outer-wrapper layout-caption-below design-layout-inline" id="yui_3_17_2_1_1509333291918_308">
-<div class="intrinsic" style="max-width:268.0px;" id="yui_3_17_2_1_1509333291918_307">
-	<div style="padding-bottom: 56.7164%; overflow: hidden;" class="image-block-wrapper   has-aspect-ratio" data-description="<p>
-		<em>Fig 10. Electrophoresis of the digested pSB1C3 vector.</em>
-	</p>
-	" id="yui_3_17_2_1_1509333291918_306">
-	<noscript>
-		<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f402b36926700aba0d0796/1509163705061/10.jpg"  alt="Fig 10. Electrophoresis of the digested pSB1C3 vector."  />
-	</noscript>
-	<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f402b36926700aba0d0796/1509163705061/10.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f402b36926700aba0d0796/1509163705061/10.jpg" data-image-dimensions="268x152" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f402b36926700aba0d0796" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 10. Electrophoresis of the digested pSB1C3 vector." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f402b36926700aba0d0796/1509163705061/10.jpg?format=300w" data-image-resolution="300w"></div>
-<div class="image-caption-wrapper">
-	<div class="image-caption">
-		<p>
-			<em>Fig 10. Electrophoresis of the digested pSB1C3 vector.</em>
-		</p>
 	</div>
-</div>
-</div>
-</div>
-</div>
-</div>
-<div class="sqs-block html-block sqs-block-html" data-block-type="2" id="block-yui_3_17_2_4_1509162483656_31152">
-<div class="sqs-block-content">
-<p>
-<!--[if gte vml 1]>
-<v:shape
- id="_x0000_i1036" type="#_x0000_t75" style='width:201pt;height:114pt'>
-<v:imagedata src="file:////Users/tianyuwang/Library/Group%20Containers/UBF8T346G9.Office/msoclip1/01/clip_image023.png"
-&nbsp; o:title=""/>
-</v:shape>
-<![endif]-->
-</p>
-<p>
-.3.3 &nbsp;&nbsp;&nbsp;&nbsp; Ligation of purified miR-21-sponge-6s and miR-21-sponge-2s fragment&nbsp; to pSB1C3 vector
-</p>
-<p>
-We ligated the purified miR-21-sponge-6s or miR-21-sponge-2s fragment to pSB1C3 vector, respectively. Then we selected the positive clones by PCR and sequencing (Fig 11 and 12).
-</p>
-</div>
-</div>
-<div class="sqs-block image-block sqs-block-image sqs-text-ready" data-block-type="5" id="block-yui_3_17_2_4_1509162483656_49246">
-<div class="sqs-block-content" id="yui_3_17_2_1_1509333291918_326">
-<div class="image-block-outer-wrapper layout-caption-below design-layout-inline" id="yui_3_17_2_1_1509333291918_325">
-<div class="intrinsic" style="max-width:560.0px;" id="yui_3_17_2_1_1509333291918_324">
-<div style="padding-bottom: 29.4643%; overflow: hidden;" class="image-block-wrapper   has-aspect-ratio" data-description="" id="yui_3_17_2_1_1509333291918_323">
-	<noscript>
-		<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f402f510952624f5425864/1509163778597/11ab.jpg"  alt="11ab.jpg"  />
-	</noscript>
-	<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f402f510952624f5425864/1509163778597/11ab.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f402f510952624f5425864/1509163778597/11ab.jpg" data-image-dimensions="560x165" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f402f510952624f5425864" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="11ab.jpg" src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f402f510952624f5425864/1509163778597/11ab.jpg?format=750w" data-image-resolution="750w"></div>
-</div>
-</div>
-</div>
-</div>
-<div class="sqs-block image-block sqs-block-image sqs-text-ready" data-block-type="5" id="block-yui_3_17_2_4_1509162483656_52024">
-<div class="sqs-block-content" id="yui_3_17_2_1_1509333291918_343">
-<div class="image-block-outer-wrapper layout-caption-below design-layout-inline" id="yui_3_17_2_1_1509333291918_342">
-<div class="intrinsic" style="max-width:696.0px;" id="yui_3_17_2_1_1509333291918_341">
-<div style="padding-bottom: 38.0747%; overflow: hidden;" class="image-block-wrapper   has-aspect-ratio" data-description="" id="yui_3_17_2_1_1509333291918_340">
-	<noscript>
-		<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403102774d1138b78c19a/1509163808883/11c.jpg"  alt="11c.jpg"  />
-	</noscript>
-	<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403102774d1138b78c19a/1509163808883/11c.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403102774d1138b78c19a/1509163808883/11c.jpg" data-image-dimensions="696x265" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f403102774d1138b78c19a" data-type="image" data-position-mode="standard" style="left: 0%; top: -0.0781863%; width: 100%; height: 100.156%; position: absolute;" alt="11c.jpg" src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403102774d1138b78c19a/1509163808883/11c.jpg?format=750w" data-image-resolution="750w"></div>
-</div>
-</div>
-</div>
-</div>
-<div class="sqs-block image-block sqs-block-image sqs-text-ready" data-block-type="5" id="block-yui_3_17_2_4_1509162483656_59772">
-<div class="sqs-block-content" id="yui_3_17_2_1_1509333291918_360">
-<div class="image-block-outer-wrapper layout-caption-below design-layout-inline" id="yui_3_17_2_1_1509333291918_359">
-<div class="intrinsic" style="max-width:259.0px;" id="yui_3_17_2_1_1509333291918_358">
-<div style="padding-bottom: 59.4595%; overflow: hidden;" class="image-block-wrapper   has-aspect-ratio" data-description="<p>
-	<em>
-		Fig 12. Electrophoresis of pSB1C3- miR-21-sponge-6s (lane 1, 2) and&amp;nbsp; pSB1C3- miR-21-sponge-2s (lane 3, 4) vector.
-	</em>
-</p>
-" id="yui_3_17_2_1_1509333291918_357">
-<noscript>
-	<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403336c31940534284984/1509163831033/12.jpg"  alt="Fig 12. Electrophoresis of pSB1C3- miR-21-sponge-6s (lane 1, 2) and  pSB1C3- miR-21-sponge-2s (lane 3, 4) vector."  />
-</noscript>
-<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403336c31940534284984/1509163831033/12.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403336c31940534284984/1509163831033/12.jpg" data-image-dimensions="259x154" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f403336c31940534284984" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 12. Electrophoresis of pSB1C3- miR-21-sponge-6s (lane 1, 2) and&nbsp; pSB1C3- miR-21-sponge-2s (lane 3, 4) vector." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403336c31940534284984/1509163831033/12.jpg?format=300w" data-image-resolution="300w"></div>
-<div class="image-caption-wrapper">
-<div class="image-caption">
-	<p>
-		<em>
-			Fig 12. Electrophoresis of pSB1C3- miR-21-sponge-6s (lane 1, 2) and&nbsp; pSB1C3- miR-21-sponge-2s (lane 3, 4) vector.
-		</em>
-	</p>
-</div>
-</div>
-</div>
-</div>
-</div>
 </div>
 <div class="sqs-block html-block sqs-block-html" data-block-type="2" id="block-yui_3_17_2_4_1509162483656_60050">
-<div class="sqs-block-content">
+	<div class="sqs-block-content">
-<p>
+		<p>
-<!--[if gte vml 1]>
+			<!--[if gte vml 1]>
-<v:shape
+			<v:shape
   id="_x0000_i1037" type="#_x0000_t75" style='width:412pt;height:122pt'>
-<v:imagedata src="file:////Users/tianyuwang/Library/Group%20Containers/UBF8T346G9.Office/msoclip1/01/clip_image025.png"
+				<v:imagedata src="file:////Users/tianyuwang/Library/Group%20Containers/UBF8T346G9.Office/msoclip1/01/clip_image025.png"
 &nbsp; o:title=""/>
-</v:shape>
+			</v:shape>
-<![endif]-->
+			<![endif]-->
-</p>
+		</p>
-<p>
+		<p>
-<!--[if gte vml 1]>
+			<!--[if gte vml 1]>
-<v:shape
+			<v:shape
   id="_x0000_i1038" type="#_x0000_t75" style='width:359pt;height:71pt'>
-<v:imagedata src="file:////Users/tianyuwang/Library/Group%20Containers/UBF8T346G9.Office/msoclip1/01/clip_image027.png"
+				<v:imagedata src="file:////Users/tianyuwang/Library/Group%20Containers/UBF8T346G9.Office/msoclip1/01/clip_image027.png"
 &nbsp; o:title=""/>
-</v:shape>
+			</v:shape>
-<![endif]-->
+			<![endif]-->
-</p>
+		</p>
-<p>
+		<p>
-<!--[if gte vml 1]>
+			<!--[if gte vml 1]>
-<v:shape
+			<v:shape
   id="图片_x0020_11" o:spid="_x0000_i1039" type="#_x0000_t75" style='width:353pt;
   height:70pt;visibility:visible'>
-<v:imagedata src="file:////Users/tianyuwang/Library/Group%20Containers/UBF8T346G9.Office/msoclip1/01/clip_image029.png"
+				<v:imagedata src="file:////Users/tianyuwang/Library/Group%20Containers/UBF8T346G9.Office/msoclip1/01/clip_image029.png"
 &nbsp; o:title=""/>
-</v:shape>
+			</v:shape>
-<![endif]-->
+			<![endif]-->
-</p>
+		</p>
-<p>
+		<p>
-<strong>1.4</strong>
+			<strong>1.4</strong>
-&nbsp;The solid plates of each plasmid constructed (Fig 13)
+			&nbsp;The solid plates of each plasmid constructed (Fig 13)
-</p>
+		</p>
-</div>
+	</div>
 </div>
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-<div class="sqs-block-content" id="yui_3_17_2_1_1509333291918_377">
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-<div class="image-block-outer-wrapper layout-caption-below design-layout-inline" id="yui_3_17_2_1_1509333291918_376">
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-<div style="padding-bottom: 73.9409%; overflow: hidden;" class="image-block-wrapper   has-aspect-ratio" data-description="<p>
+				<div style="padding-bottom: 73.9409%; overflow: hidden;" class="image-block-wrapper   has-aspect-ratio" data-description="<p>
-<em>Fig 13 Solid plates of five plasmids</em>
+					<em>Fig 13 Solid plates of five plasmids</em>
-</p>
+				</p>
-" id="yui_3_17_2_1_1509333291918_374">
+				" id="yui_3_17_2_1_1509572305608_374">
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-<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403756c31940534284d6b/1509163900773/13.jpg"  alt="Fig 13 Solid plates of five plasmids"  />
+					<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403756c31940534284d6b/1509163900773/13.jpg"  alt="Fig 13 Solid plates of five plasmids"  />
-</noscript>
+				</noscript>
-<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403756c31940534284d6b/1509163900773/13.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403756c31940534284d6b/1509163900773/13.jpg" data-image-dimensions="779x576" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f403756c31940534284d6b" data-type="image" data-position-mode="standard" style="left: 0%; top: -0.0323577%; width: 100%; height: 100.065%; position: absolute;" alt="Fig 13 Solid plates of five plasmids" src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403756c31940534284d6b/1509163900773/13.jpg?format=750w" data-image-resolution="750w"></div>
+				<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403756c31940534284d6b/1509163900773/13.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403756c31940534284d6b/1509163900773/13.jpg" data-image-dimensions="779x576" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f403756c31940534284d6b" data-type="image" data-position-mode="standard" style="left: 0%; top: -0.0323577%; width: 100%; height: 100.065%; position: absolute;" alt="Fig 13 Solid plates of five plasmids" src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403756c31940534284d6b/1509163900773/13.jpg?format=750w" data-image-resolution="750w"></div>
-<div class="image-caption-wrapper">
+			<div class="image-caption-wrapper">
-<div class="image-caption">
+				<div class="image-caption">
-<p>
+					<p>
-	<em>Fig 13 Solid plates of five plasmids</em>
+						<em>Fig 13 Solid plates of five plasmids</em>
-</p>
+					</p>
-</div>
+				</div>
-</div>
+			</div>
-</div>
+		</div>
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+	</div>
 </div>
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 <div class="sqs-block html-block sqs-block-html" data-block-type="2" id="block-yui_3_17_2_4_1509162483656_76104">
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+			<v:imagedata src="file:////Users/tianyuwang/Library/Group%20Containers/UBF8T346G9.Office/msoclip1/01/clip_image031.png"
 &nbsp; o:title=""/>
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-</p>
+	</p>
-<p>
+	<p>
-<strong>2. The effect of miR-21 sponges in cells</strong>
+		<strong>2. The effect of miR-21 sponges in cells</strong>
-</p>
+	</p>
-<p>
+	<p>
-&nbsp;To detect the validity of our detection device, we transfected pEGFP-miR-21-sponge-6s or pEGFP-miR-21-sponge-2s (0.25ug /well) with pHAGE-puro (0.25ug /well, as negative control) into 293T cells in 24 well-plate, respectively. At same times, we transfected pEGFP-miR-21-sponge-6s or pEGFP-miR-21-sponge-2s (0.25ug/well) with pHAGE-pre-miR-21 (0.25ug/well) into 293T cells. Then the GFP fluorescence was observed under fluorescence microscopy (Fig 14 A,B,C,D). The result suggested our detection device worked very well, and overexpression of miR-21 can decrease the fluorescence intensity of pEGFP-miR-21-sponge-6s and pEGFP-miR-21-sponge-2s.
+		&nbsp;To detect the validity of our detection device, we transfected pEGFP-miR-21-sponge-6s or pEGFP-miR-21-sponge-2s (0.25ug /well) with pHAGE-puro (0.25ug /well, as negative control) into 293T cells in 24 well-plate, respectively. At same times, we transfected pEGFP-miR-21-sponge-6s or pEGFP-miR-21-sponge-2s (0.25ug/well) with pHAGE-pre-miR-21 (0.25ug/well) into 293T cells. Then the GFP fluorescence was observed under fluorescence microscopy (Fig 14 A,B,C,D). The result suggested our detection device worked very well, and overexpression of miR-21 can decrease the fluorescence intensity of pEGFP-miR-21-sponge-6s and pEGFP-miR-21-sponge-2s.
-</p>
+	</p>
-<p>
+	<p>
-And we also detected the fluorescence of GFP after transfection pEGFP-miR-21-sponge-6s or pEGFP- miR-21-sponge-2s with or without pHAGE-pre-miR-21 for 24 h by plate reader (SpectraMax i3), and observed that endogenous miR-21 in cells decreased the fluorescence of GFP (Fig 15). The fluorescence of GFP in cells transfect with pEGFP-miR-21-sponge-6s was lower in that of pEGFP-miR-21-sponge-2s, because pEGFP-miR-21-sponge-6s contains six miR-21 binding sites. These results suggested that sponges with six binding sites produced stronger derepressiveeffects than sponges with two binding sites.
+		And we also detected the fluorescence of GFP after transfection pEGFP-miR-21-sponge-6s or pEGFP- miR-21-sponge-2s with or without pHAGE-pre-miR-21 for 24 h by plate reader (SpectraMax i3), and observed that endogenous miR-21 in cells decreased the fluorescence of GFP (Fig 15). The fluorescence of GFP in cells transfect with pEGFP-miR-21-sponge-6s was lower in that of pEGFP-miR-21-sponge-2s, because pEGFP-miR-21-sponge-6s contains six miR-21 binding sites. These results suggested that sponges with six binding sites produced stronger derepressiveeffects than sponges with two binding sites.
-</p>
+	</p>
 </div>
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+			<div style="padding-bottom: 99.7006%; overflow: hidden;" class="image-block-wrapper   has-aspect-ratio" data-description="<p>
-<em>
+				<em>
-Fig 14. &amp;nbsp;The images of 293T cells transfected different plasmids A. pEGFP- miR-21-sponge-6s and pHAGE-puro co-transfected. B. pEGFP-miR-21-sponge-2s and pHAGE-puro co-transfected. C. pEGFP-miR-21-sponge-6s and pHAGE-pre-miR-21 co-transfected. D. pEGFP- miR-21-sponge-2s and pHAGE-pre-miR-21 co-transfected, Bar = 100mm .
+					Fig 14. &amp;nbsp;The images of 293T cells transfected different plasmids A. pEGFP- miR-21-sponge-6s and pHAGE-puro co-transfected. B. pEGFP-miR-21-sponge-2s and pHAGE-puro co-transfected. C. pEGFP-miR-21-sponge-6s and pHAGE-pre-miR-21 co-transfected. D. pEGFP- miR-21-sponge-2s and pHAGE-pre-miR-21 co-transfected, Bar = 100mm .
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-<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403bce2c483f7f6cbd90a/1509164327754/14.jpg"  alt="Fig 14.  The images of 293T cells transfected different plasmids A. pEGFP- miR-21-sponge-6s and pHAGE-puro co-transfected. B. pEGFP-miR-21-sponge-2s and pHAGE-puro co-transfected. C. pEGFP-miR-21-sponge-6s and pHAGE-pre-miR-21 co-transfected. D. pEGFP- miR-21-sponge-2s and pHAGE-pre-miR-21 co-transfected, Bar = 100mm ."  />
+				<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403bce2c483f7f6cbd90a/1509164327754/14.jpg"  alt="Fig 14.  The images of 293T cells transfected different plasmids A. pEGFP- miR-21-sponge-6s and pHAGE-puro co-transfected. B. pEGFP-miR-21-sponge-2s and pHAGE-puro co-transfected. C. pEGFP-miR-21-sponge-6s and pHAGE-pre-miR-21 co-transfected. D. pEGFP- miR-21-sponge-2s and pHAGE-pre-miR-21 co-transfected, Bar = 100mm ."  />
-</noscript>
+			</noscript>
-<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403bce2c483f7f6cbd90a/1509164327754/14.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403bce2c483f7f6cbd90a/1509164327754/14.jpg" data-image-dimensions="334x333" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f403bce2c483f7f6cbd90a" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 14. &nbsp;The images of 293T cells transfected different plasmids A. pEGFP- miR-21-sponge-6s and pHAGE-puro co-transfected. B. pEGFP-miR-21-sponge-2s and pHAGE-puro co-transfected. C. pEGFP-miR-21-sponge-6s and pHAGE-pre-miR-21 co-transfected. D. pEGFP- miR-21-sponge-2s and pHAGE-pre-miR-21 co-transfected, Bar = 100mm ." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403bce2c483f7f6cbd90a/1509164327754/14.jpg?format=500w" data-image-resolution="500w"></div>
+			<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403bce2c483f7f6cbd90a/1509164327754/14.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403bce2c483f7f6cbd90a/1509164327754/14.jpg" data-image-dimensions="334x333" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f403bce2c483f7f6cbd90a" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 14. &nbsp;The images of 293T cells transfected different plasmids A. pEGFP- miR-21-sponge-6s and pHAGE-puro co-transfected. B. pEGFP-miR-21-sponge-2s and pHAGE-puro co-transfected. C. pEGFP-miR-21-sponge-6s and pHAGE-pre-miR-21 co-transfected. D. pEGFP- miR-21-sponge-2s and pHAGE-pre-miR-21 co-transfected, Bar = 100mm ." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403bce2c483f7f6cbd90a/1509164327754/14.jpg?format=500w" data-image-resolution="500w"></div>
-<div class="image-caption-wrapper">
+		<div class="image-caption-wrapper">
-<div class="image-caption">
+			<div class="image-caption">
-<p>
+				<p>
-<em>
+					<em>
-	Fig 14. &nbsp;The images of 293T cells transfected different plasmids A. pEGFP- miR-21-sponge-6s and pHAGE-puro co-transfected. B. pEGFP-miR-21-sponge-2s and pHAGE-puro co-transfected. C. pEGFP-miR-21-sponge-6s and pHAGE-pre-miR-21 co-transfected. D. pEGFP- miR-21-sponge-2s and pHAGE-pre-miR-21 co-transfected, Bar = 100mm .
+						Fig 14. &nbsp;The images of 293T cells transfected different plasmids A. pEGFP- miR-21-sponge-6s and pHAGE-puro co-transfected. B. pEGFP-miR-21-sponge-2s and pHAGE-puro co-transfected. C. pEGFP-miR-21-sponge-6s and pHAGE-pre-miR-21 co-transfected. D. pEGFP- miR-21-sponge-2s and pHAGE-pre-miR-21 co-transfected, Bar = 100mm .
-</em>
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+		<div style="padding-bottom: 57.1717%; overflow: hidden;" class="image-block-wrapper   has-aspect-ratio" data-description="<p>
-<em>
+			<em>
-Fig 15. The fluorescence of GFP after co-transfection pEGFP-miR-21-sponge-6s or pEGFP- miR-21-sponge-2s with or without pHAGE-pre-miR-21 .
+				Fig 15. The fluorescence of GFP after co-transfection pEGFP-miR-21-sponge-6s or pEGFP- miR-21-sponge-2s with or without pHAGE-pre-miR-21 .
-</em>
+			</em>
-</p>
+		</p>
-" id="yui_3_17_2_1_1509333291918_408">
+		" id="yui_3_17_2_1_1509572305608_408">
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+			<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403fc27ef2d09a2192ecf/1509164034255/15.jpg"  alt="Fig 15. The fluorescence of GFP after co-transfection pEGFP-miR-21-sponge-6s or pEGFP- miR-21-sponge-2s with or without pHAGE-pre-miR-21 ."  />
-</noscript>
+		</noscript>
-<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403fc27ef2d09a2192ecf/1509164034255/15.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403fc27ef2d09a2192ecf/1509164034255/15.jpg" data-image-dimensions="495x283" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f403fc27ef2d09a2192ecf" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 15. The fluorescence of GFP after co-transfection pEGFP-miR-21-sponge-6s or pEGFP- miR-21-sponge-2s with or without pHAGE-pre-miR-21 ." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403fc27ef2d09a2192ecf/1509164034255/15.jpg?format=500w" data-image-resolution="500w"></div>
+		<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403fc27ef2d09a2192ecf/1509164034255/15.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403fc27ef2d09a2192ecf/1509164034255/15.jpg" data-image-dimensions="495x283" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f403fc27ef2d09a2192ecf" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 15. The fluorescence of GFP after co-transfection pEGFP-miR-21-sponge-6s or pEGFP- miR-21-sponge-2s with or without pHAGE-pre-miR-21 ." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f403fc27ef2d09a2192ecf/1509164034255/15.jpg?format=500w" data-image-resolution="500w"></div>
-<div class="image-caption-wrapper">
+	<div class="image-caption-wrapper">
-<div class="image-caption">
+		<div class="image-caption">
-<p>
+			<p>
-<em>
+				<em>
-Fig 15. The fluorescence of GFP after co-transfection pEGFP-miR-21-sponge-6s or pEGFP- miR-21-sponge-2s with or without pHAGE-pre-miR-21 .
+					Fig 15. The fluorescence of GFP after co-transfection pEGFP-miR-21-sponge-6s or pEGFP- miR-21-sponge-2s with or without pHAGE-pre-miR-21 .
-</em>
+				</em>
-</p>
+			</p>
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 .1 The effect of miR-21 sponges as a monitor to detect the expression of miR-21
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 <strong>
-<em>
+	<em>
-&nbsp;&nbsp;&nbsp;&nbsp; copies/ul= (6.02×1023)×(plasmids concentrations ng/ul×10-9)/(DNA length×660)
+		&nbsp;&nbsp;&nbsp;&nbsp; copies/ul= (6.02×1023)×(plasmids concentrations ng/ul×10-9)/(DNA length×660)
-</em>
+	</em>
 </strong>
 </p>
 <p>
-The standard curve of miR-21-sponge-6s and miR-21-sponge-2s were made by EXCEL, respectively (Fig 16 and 17). We find that when miR-21 copies changed the slope of the standard curve of miR-21-sponge-6s was greater than miR-21-sponge-2s which indicated miR-21-sponge-6s is more sensitive than miR-21-sponge-2s. The correlation coefficient (R2 value) of miR-21-sponge-6s was 0.9993 and R2 value of miR-21-sponge-6s was 0.8995.
+The standard curve of miR-21-sponge-6s and miR-21-sponge-2s were made by EXCEL, respectively (Fig 16 and 17). We find that when miR-21 copies changed the slope of the standard curve of miR-21-sponge-2s was greater than miR-21-sponge-6s which indicated miR-21-sponge-2s is more sensitive than miR-21-sponge6s. However, the correlation coefficient (R2 value) of miR-21-sponge-6s was 0.9993 and R2 value of miR-21-sponge-6s was 0.8995. The R2 value of miR-21-sponge-6s is better than miR-21-sponge-2s.
 </p>
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+	<div style="padding-bottom: 51.1222%; overflow: hidden;" class="image-block-wrapper   has-aspect-ratio" data-description="<p>
-<em>
+		<em>
-Fig 16. The effect of pEGFP-miR-21-sponge-6s or pEGFP-miR-21-sponge-2s on the correlative between fluorescence value of GFP and copy numbers of miR-21.
+			Fig 16. The effect of pEGFP-miR-21-sponge-6s or pEGFP-miR-21-sponge-2s on the correlative between fluorescence value of GFP and copy numbers of miR-21.
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+		</em>
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+		<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f7f4fd8e7b0fa62a247fc9/1509422334420/aaa.jpg"  alt="Fig 16. The effect of pEGFP-miR-21-sponge-6s or pEGFP-miR-21-sponge-2s on the correlative between fluorescence value of GFP and copy numbers of miR-21."  />
-</noscript>
+	</noscript>
-<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f4046941920221548bfb63/1509164141807/16.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f4046941920221548bfb63/1509164141807/16.jpg" data-image-dimensions="502x256" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f4046941920221548bfb63" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 16. The effect of pEGFP-miR-21-sponge-6s or pEGFP-miR-21-sponge-2s on the correlative between fluorescence value of GFP and copy numbers of miR-21." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f4046941920221548bfb63/1509164141807/16.jpg?format=750w" data-image-resolution="750w"></div>
+	<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f7f4fd8e7b0fa62a247fc9/1509422334420/aaa.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f7f4fd8e7b0fa62a247fc9/1509422334420/aaa.jpg" data-image-dimensions="401x205" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f7f4fd8e7b0fa62a247fc9" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 16. The effect of pEGFP-miR-21-sponge-6s or pEGFP-miR-21-sponge-2s on the correlative between fluorescence value of GFP and copy numbers of miR-21." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f7f4fd8e7b0fa62a247fc9/1509422334420/aaa.jpg?format=500w" data-image-resolution="500w"></div>
 <div class="image-caption-wrapper">
-<div class="image-caption">
+	<div class="image-caption">
-<p>
+		<p>
-<em>
+			<em>
-Fig 16. The effect of pEGFP-miR-21-sponge-6s or pEGFP-miR-21-sponge-2s on the correlative between fluorescence value of GFP and copy numbers of miR-21.
+				Fig 16. The effect of pEGFP-miR-21-sponge-6s or pEGFP-miR-21-sponge-2s on the correlative between fluorescence value of GFP and copy numbers of miR-21.
-</em>
+			</em>
-</p>
+		</p>
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+	</div>
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+<div style="padding-bottom: 90.8642%; overflow: hidden;" class="image-block-wrapper   has-aspect-ratio" data-description="<p>
-<em>
+	<em>
-Fig 17. The standard curve of miR-21-sponge-6s and miR-21-sponge-2s.
+		Fig 17. The standard curve of miR-21-sponge-6s and miR-21-sponge-2s.
-</em>
+	</em>
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-" id="yui_3_17_2_1_1509333291918_459">
+" id="yui_3_17_2_1_1509572305608_442">
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-<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f404aeec212d295ba9cd98/1509164255600/17%282%29.jpg"  alt="Fig 17. The standard curve of miR-21-sponge-6s and miR-21-sponge-2s."  />
+	<img src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f7ebc06926704f3555ecd8/1509419971690/17%282%29.jpg"  alt="Fig 17. The standard curve of miR-21-sponge-6s and miR-21-sponge-2s."  />
 </noscript>
-<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f404aeec212d295ba9cd98/1509164255600/17%282%29.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f404aeec212d295ba9cd98/1509164255600/17%282%29.jpg" data-image-dimensions="496x208" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f404aeec212d295ba9cd98" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 17. The standard curve of miR-21-sponge-6s and miR-21-sponge-2s." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f404aeec212d295ba9cd98/1509164255600/17%282%29.jpg?format=500w" data-image-resolution="500w"></div>
+<img class="thumb-image loaded" data-src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f7ebc06926704f3555ecd8/1509419971690/17%282%29.jpg" data-image="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f7ebc06926704f3555ecd8/1509419971690/17%282%29.jpg" data-image-dimensions="405x368" data-image-focal-point="0.5,0.5" data-load="false" data-image-id="59f7ebc06926704f3555ecd8" data-type="image" data-position-mode="standard" style="left: 0%; top: 0%; width: 100%; height: 100%; position: absolute;" alt="Fig 17. The standard curve of miR-21-sponge-6s and miR-21-sponge-2s." src="https://static1.squarespace.com/static/598d6f47ff7c50fa2d55f1dc/t/59f7ebc06926704f3555ecd8/1509419971690/17%282%29.jpg?format=500w" data-image-resolution="500w"></div>
 <div class="image-caption-wrapper">
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-<p>
+	<p>
-<em>
+		<em>
-Fig 17. The standard curve of miR-21-sponge-6s and miR-21-sponge-2s.
+			Fig 17. The standard curve of miR-21-sponge-6s and miR-21-sponge-2s.
-</em>
+		</em>
-</p>
+	</p>
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-<strong>Conclusions and perspective</strong>
+<strong>Conclusions and perspectives</strong>
-<strong>s</strong>
+</p>
+<p>
+The results reported here demonstrate that miRNA sponge may be a monitor to detect the expression of miRNA in cells. And we also suggested the slope of the standard curve of miR-21-sponge-2s is better than miR-21-sponge-6s, suggesting the more sensitive of miR-21-sponge-2s as a monitor. Our results for the first time to evaluate the possibility of miRNA sponge for detecting miRNA and provide a new method for diagnosis miRNA in the future. However, there are still lots of issues relative to this new method that we need to consider. Firstly, we should evaluate the sensitivity and specificity of this technique by compare with other method including real-time PCR. Next, whether miRNA sponge contained two binding sites is the best monitor should be considered and tested. More strategies should be thought to improve the sensitivity and specificity of this technique by synthetic biology.
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-The results reported here demonstrate that miRNA sponge may be a moniter to detect the expression of miRNA in cells. And we also suggested the slope and the correlation coefficient of miR-21-sponge-6s is better than miR-21-sponge-2s. Our results for the first time to evaluate the possibility of miRNA sponge for detecting miRNA and provide a new method for diagnosis miRNA in the future. However, there are still lots of issues relative to this new method that we need to consider. Firstly, we should evaluate the sensitivity and specificity of this technique by compare with other method including real-time PCR. Next, whether miRNA sponge contained six binding sites is the best monitor should be considered.&nbsp; More strategies should be thought to improve the sensitivity and specificity of this technique by synthetic biology.
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+		<use class="sqs-use--mask" xlink:href="#instagram-mask"></use>
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+	<svg class="sqs-svg-icon--social" viewBox="0 0 64 64">
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+		<use class="sqs-use--icon" xlink:href="#email-icon"></use>
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+		<use class="sqs-use--mask" xlink:href="#email-mask"></use>
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+	<svg class="sqs-svg-icon--social" viewBox="0 0 64 64">
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Difference between revisions of "Team:Worldshaper-Wuhan/Results"

Latest revision as of 00:45, 2 November 2017

Result