Biomod/2014/OhioMOD/experimentnotes

content{margin-left:0;}

.navbar{ top:8.5px; position:fixed; width:96.8%; } /*clearing the side openwetware panels*/ .firstHeading, #column-one, #p-bookmarks, #p-history , .portlet{ display:none; }

bodyContent/*clearing the side openwetware panels

.firstHeading, #column-one, #p-bookmarks, #p-history , .portlet{ display:none; }

content#bodyContent, {

/*display:none;*/ background-color:#FFFFFF; }

OSUfooter{

clear:both; }

footer{display: none;}

/*Done clearing*/

</style>

</head>

<div class="navbar navbar-default">

 <div class="navbar-header">
   <button type="button" class="navbar-toggle" data-toggle="collapse" data-target=".navbar-responsive-collapse">
     <span class="icon-bar"></span>
     <span class="icon-bar"></span>
     <span class="icon-bar"></span>
   </button>
   <a class="navbar-brand" href="http://openwetware.org/wiki/Biomod/2014/OhioMOD">OhioMOD</a>
 </div>
 <div class="navbar-collapse collapse navbar-responsive-collapse">
   <ul class="nav navbar-nav">
     <li ><a href="http://openwetware.org/wiki/Biomod/2014/OhioMOD">Home</a></li>
     <li><a href="http://openwetware.org/wiki/Biomod/2014/OhioMOD/project">Background</a></li>

<li><a href="http://openwetware.org/wiki/Biomod/2014/OhioMOD/results">Project and Results</a></li>

       <a href="#" class="dropdown-toggle" data-toggle="dropdown">Materials and Methods <b class="caret"></b></a>
       <ul class="dropdown-menu">
         <li><a href="http://openwetware.org/wiki/Biomod/2014/OhioMOD/protocols">Protocols</a></li>
         <li><a href="http://openwetware.org/wiki/Biomod/2014/OhioMOD/equipment">Equipment</a></li>
         <li class="divider"></li>
         <li class="dropdown-header">Lab Notebook</li>
         <li><a href="http://openwetware.org/wiki/Biomod/2014/OhioMOD/LabNotebook">Experiment Schedule</a></li>
         <li><a href="http://openwetware.org/wiki/Biomod/2014/OhioMOD/experimentnotes">Experiment Notes</a></li>
       </ul>
     </li>

<li><a href="http://openwetware.org/wiki/Biomod/2014/OhioMOD/team">Team</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2014/OhioMOD/sponsors">Acknowledgement</a></li> <li><a href="http://www.giveto.osu.edu/igive/OnlineGiving/fund_results.aspx?fund=314139" target="_blank">Give To OhioMOD</a></li>

   </ul>
 </div>

</div>

</html> <html> <head> <style> center {padding-bottom:50px;}

ExperimentNotes{

 width: 700px ;
 margin-left: auto ;
 margin-right: auto ;
 margin-bottom: 50px;

}

list2

{

text-indent:50px;

}

/*fixed icon code start*/

fixedbutton {

   position: fixed;
   bottom: 53px;
   right: 10px;

} /*fixed icon code end*/

</style>

</head> <body> <div id="ExperimentNotes">

<h1>Experiment Notes</h1>

  <TR>
     <TH COLSPAN="2"><strong>Table of Contents</strong>
     </TH>
  </TR>

     <TR>
     <TD>

<A HREF="#scroll1">1. Cellular Uptake Experiment</A> <A HREF="#scroll2">2. miR-21 Sequestration Experiment</A> <A NAME="scroll1"></A><A HREF="#scroll3">3. Cell Viability Experiment</A> <A HREF="#scroll4">4. PTEN Protein Expression</A> <A HREF="#scroll5">5. PTEN mRNA Experiment</A>

</TD>

  </TR>

</TABLE>

<div id="1"> <h3>1. Cellular Uptake Experiment</h3> Treatments:

1. Cells only: Lysotracker stained cells only. 2. Branch (Br scr): Lysotracker stained cells with TOPRO3 stained branch scrambled overhang. 3. Block O (BO scr): Lysotracker stained cells with TOPRO3 stained Block O scrambled overhangs.

Procedure: 1. Staining of structures 1.1 Measure the concentration of structures and concentration of dye needed to stain 50 uL of structures</ul> 1.2 Add correct amount of dye to 50 uL of structure and incubate overnight 2. Staining of cells 2.1 Combine 0.1 uL of 1 mM Lysotracker with 1 mL of Human RPMI media to get final concentration of 100 nM. Incubate at 37 C. 2.2 Count cells. Aliquot 3 samples with ~75,000 cells in each. 2.3 Wash cells with PBS. Resuspend in 200 uL of human media containing Lysotracker dye. Incubate for 1.5 hrs. 3. Structure addition 3.1 After incubation, wash cells with PBS 3.2 Resuspend cells in clear media with 10% FBS. a 175 uL clear media and 25 uL FBS. 3.3 Plate cells in 8 well imaging plate. Add 50 uL of structures to corresponding plates. a. Sample 1 will be unstained cells only (CO Add 50 uL of TAE buffer b. Sample 2 will be stained cells with branch stained with TOPRO. structure concentration: 0.2 nM c. Sample 3 will be stained cells with Block O stained with TOPRO. structure concentration: 0.2 nM 4. TIRF imaging 4.1 Immediately image under TIRF to get t=0 data. After imaging, reincubate for 4 hrs >4.2 Image again to obtain data for t=4 hours</ul>

Results:

t = 0 • Cells only:

<figure> <img src="http://openwetware.org/images/4/48/Exptfig1.png"height="217" width="624"/> <figcaption>Figure 1: Cells stained with Lysotracker only.</figcaption> </figure>

The cells only images at time 0 showed ideal Lysotracker staining, with very little fluorescent signal in the 640 channel. • Branch

<figure> <img src="http://openwetware.org/images/9/9c/Expfig2.png"height="209" width="624"/> <figcaption>Figure 2: Stained cells incubated with stained branch structures.</figcaption> </figure>

</brThe images of cells with branch structures exhibits a strong signal in both the 488 and the 640 channel, confirming the successful staining by both the Lysotracker and the TOPRO3. However, while the Lysotracker signal is present in both cells, the TOPRO3 signal is present only in the cell with a highly granular interior, suggesting a cell nearing death. • Block O

<figure> <img src="http://openwetware.org/images/f/fd/Expfig3.png"height="206" width="624"/> <figcaption>Figure 3: Stained cells with stained Block O structures.</figcaption> </figure>

The cells incubated with Block O showed a weaker Lysotracker staining than the cells with branch. However, the stain was still discernible. As in the branch sample, there was a strong fluorescent signal in the 640 channel which corresponds to stained structures. However, as with the branch sample, the fluorescence originates from dead cell debris rather than any live cells. There was no detectable co-localization of the two signals in any of the samples imaged at t = 0. t = 4 • Cells only

<figure> <img src="http://openwetware.org/images/b/bb/Expfig4.png"height="206" width="620"/> <figcaption>Figure 4: Cells stained with Lysotracker only.</figcaption> </figure>

The cells imaged after the 4 hour incubation displayed a weaker Lysotracker signal than at t = 0. There were weak signals in the 640 channel as well. However, due to the low intensities and high backgrounds, those are most likely residual fluorescence from the dye rather than something extraneous. • Branch

<figure> <img src="http://openwetware.org/images/6/6e/Expfig5.png"height="420" width="624"/> <figcaption>Figure 5: Stained cells incubated with stained branch structures.</figcaption> </figure>

The fluorescent signal from the Lysotracker, while not as strong as at t = 0, was still within ideal intensity. There were strong signals in the 640 channel as well, and the signals originated within viable cells. There were multiple points of co-localization between the 488 and 640 channels. • Block O <figure> <img src="http://openwetware.org/images/9/9a/Expfig6.png"height="423" width="622"/> <figcaption>Figure 6: Stained cells with stained Block O structures.</figcaption> </figure> <figure> <img src="http://openwetware.org/images/7/7e/Expfig7.png"height="424" width="624"/> <figcaption>Figure 7: Stained cells with Block O structures showing structures that were not yet uptaken.</figcaption> </figure>

The images for Block O at t = 4 shared many similar characteristics to those of for the branch, including multiple instances of co-localization between the two channels. However, in certain cases, a strong fluorescent signal was observed in the 640 channel located at the very edge of the cell membrane. This was not observed in any samples containing the branch structure. Discussion: In previous versions of the experiment, high cell concentration has always presented a problem. After attempting the experiment with 250,000 and 100,000 cells, 75,000 cells were used for this experiment. As a result, the cell concentration was ideal and single cells could be analyzed without interference from debris. However, the distribution of cells was not consistent, as there were spots of heavy density and spots where no cells were present. Cell viability was also better than previous experiments, possibly due to the lower cell concentration, and possibly due to an ideal concentration of FBS.

However, in cells where uptake was observed, the cell morphology was less than ideal. Cells displayed a non-circular membrane and granular cytoplasm, signs of an ailing cell. Since structures used did not contain miR-21 complementary overhangs, this could not have been due to the sequestration of miRs. Therefore, either the act of uptake caused the cells to lose ideal morphology, or structures are preferentially uptaken in ailing cells.

Many instances of dye colocalization, and therefore uptake, were observed in both the Block O and Branch treatments. However, more examples of uptake were observed in the Branch treatment. In addition, in the Block O treatment, there were instances where, after 4 hours of incubation, there were structures visible lining the membrane, perhaps in the process of being uptaken. While this is preliminary, the combination of fewer concrete instances of uptake and presence of structures still attempting to be uptaken hints that the Block O structures may not be as preferentially uptaken as the Branch structures, or may be uptaken at a slower rate. Quantitative analysis of uptake must be conducted to draw any conclusions on that matter. For future experiments, overnight time lapse experiments may provide further insight on the mechanism and rate of uptake. The cells could also be nucleofected with fluorescent miR-21 to highlight the mechanism of miR sequestration and combine both structure uptake and cellular uptake in one experiment<A NAME="scroll2"></A>.

</div>

<div id="2"> <h3>2. miR-21 Sequestration Experiment</h3> Treatments Branch

<figure> <img src="http://openwetware.org/images/2/25/2.1.png"height="52" width="460"/> </figure> Procedure • Calculate fluorescent miR-21 volume required to obtain the concentration ratios used for the treatment. Add miR-21 to 6 uL of structure and bring up volume to 18 uL with dH¬¬2O. o Structure concentration: 1 nM o miR-21 concentration: 100 nM o miR-21 volumes; <figure> <img src="http://openwetware.org/images/4/4d/2.2.png"height="88" width="462"/> </figure> • Combine structures, miR-21, and dH2O and incubate at 37 C for 30 min while shaking. • Prepare an agarose gel without EtBr according to protocol. • After incubation, load 18 uL of samples into gel and run at 70 V for 2 hours. o As controls, load 1 kb.p. DNA ladder, 7249 scaffold (branch), 8064 scaffold (Block O), and fluorescent miR-21 only. • Remove gel from apparatus and image using a laser scanner capable of fluorescence detection. • After fluorescent imaging, post-stain gel using SYBR Gold solution for 40 minutes and image. Results <figure> <img src="http://openwetware.org/images/b/bc/2.3.png"height="258" width="484"/> <figcaption>Figure 1: agarose gel image taken using bulk fluorescence.</figcaption> </figure> The miR-21 used for this experiment were tagged with the Atto 488 fluorescent dye. The image was taken using the Typhoon FLA 7000 gel imager. <figure>

<img src="http://openwetware.org/images/3/31/2.4.png"height="90" width="422"/> <figcaption>Figure 2: agarose gel image taken after staining with SYBR gold.</figcaption> </figure>

The gel was stained in 10,000x SYBR Gold bath for 40 minutes before imaging. Discussion

This experiment was designed to test the hypothesis that the complementary overhangs on the structures would successfully be able to bind to miR-21. It was also hypothesized that the structures with scrambled overhangs would not be able to sequester any miR-21 from solution. As figure 1 shows, there was no detectable fluorescence in the structure band in the lane with miR-21 and structures with scrambled overhangs, thus reinforcing the idea that the complementary overhangs are what sequesters the miR from solution.

By comparing figures 1 and 2, it is possible to conclude that, in samples containing both structures and fluorescent miR-21, the miR-21 fluorescence is colocalized with the structure bands in the gel. This shows that the structures were indeed able to remove the miR-21 from the solution.

In addition, the images also allow for conclusions to be drawn about the efficiency of sequestration. Figure 1 shows very little fluorescence beyond the structure band in the lanes with 0.33x, 0.5x and 1x samples. However, from the 2x lane onwards, there is a trend of increasing fluorescent signal beyond the structure band, suggesting that excess miR-21 is moving further along the gel. Since the increase in excess miR began after the 1x concentration, it can be concluded that the structure was able to sequester quantitative amounts of miR from the solution.

In the future, the miR sequestration experiment should be conducted with the Block O structures as well as the branch. While the experiment shows the ability of branch structures in aqueous solutions, future experiments should test for sequestration in solutions analogous to the interior of the cell, such as in cell lysate. Another avenue to test both cellular uptake and miR-21 uptake would be to nucleofect cells with fluorescent miR-21, incubate the cells with fluorescent structures, and then image them using fluorescence microscopy<A NAME="scroll3"></A>. </div>

<div id="3"> <h3>3. Cell Viability Experiment</h3> Treatments: ● Cells only: Cells incubated in media and TAE buffer. ● Branch: Cells incubated in media with branch scrambled and branch miR structures in TAE buffer. ● Block-O: Cells incubated in media with block-o scrambled and bloc-o miR structures in TAE buffer.

Procedure: ● Cell Preparation: ○ Count cells using hemocytometer and trypan blue dye ○ Separate 2*105 cells for each sample ○ Wash cells twice with PBS and resuspend in warm media ● Structure Preparation: ○ Dilute structures to desired concentration in TAE with MgCl2 ● Structure Addition: ○ Add cells in media to plate for incubation ○ Add structures to wells ○ Incubate for desired time ● Staining: ○ Add poly-L-lysine to wells in imaging plate and allow it to sit for 15 minutes ○ Remove poly-L-lysine and wash wells with PBS ○ Transfer cells from incubation plate to imaging plate ○ Add 1.75uL of Propidium Iodide (PI) to each sample for every 100 uL of solution ○ Allow the samples to sit, covered from light, for 15 minutes ○ Image the samples using the 488 nm laser

Results: time = 24 hours ● Cells only: INSERT IMAGE HERE

The cells only control showed viability of around 85%. Shown above are some examples of images taken in DIC and 488 nm.

● Branch with miR-21 complement overhangs: INSERT IMAGE HERE

The cells showed about 50% viability when treated with .5 nM branch structures with miR complement overhangs, a marked decrease in viability. Overall, there is much more fluorescence and cell morphology supports the decreased viability.

● Branch with scrambled overhangs: INSERT IMAGE HERE

These cells show viability around 80%, close to that exhibited by the cells only control. Their morphology overall supports the lower fluorescence when compared to the miR complement sample. <img src="http://openwetware.org/images/1/10/3.1.png"height="280" width="462"/> <figcaption>Figure 1. Comparison of cell viability after 24 hour incubation normalized to cells only control</figcaption> </figure> Discussion:

Previously, higher quantities of cells had been studied for viability. However, these trials resulted in crowded images that were impossible to analyze because so many cells were in different focal planes. Low cell viability was also an issue in all samples. 2*105 cells were used because that resulted in reasonable overall viability and clear images. Propidium iodide staining also had to be optimized. Low concentrations yielded very little signal, while overstaining resulted in saturated images that gave little useful information.

The images shown above are representative of the images in general. Through counting, marked decreases in viability were observed in the cells treated with the miR complement branch structure at 24 hours. Other qualitative data supports this conclusion. The morphology of the miR complement cells showed that the cells were generally unhealthy, showing irregular shape and granularity of the cytoplasm. These characteristics were not nearly as common in the under the other conditions. The histogram for signal intensity of each set of images also suggests that there was a much higher signal in the miR complement samples. The 488 signal peaked at a higher intensity and continued to higher intensities in the miR samples, while the other samples showed lower peaks and no signal at higher intensities.

Future experiments should include more time points, primarily, along with varying structure concentrations. Higher structure concentrations should yield a greater decrease in cell viability. Data at other timepoints would be useful if correlated with data on protein expression and uptake<A NAME="scroll4"></A>. </div>

<div id="4"> <h3>4. PTEN Protein Expression</h3> Treatments: <figure> <img src="http://openwetware.org/images/f/fe/1_treattable.png"height="174" width="434"/> </figure> Procedure: • Cell preparation and incubation: o Count cells and aliquot ~1 million cells per sample. o Wash cells twice and resuspend in 900 uL of cell media. o For cells only samples, add 100 uL of TAE buffer. For other samples, add 100 uL of 1 nM structures to the corresponding sample. o Plate samples in 24 well plate and incubate at 37 C for the desired timepoint (24, 48 or 72 hrs). • Protein extraction o After incubation, wash cells twice in PBS, and resuspend in 100 uL Protein Lysis buffer. Mix vigorously by pipetting. • Lysis buffer preparation: add PMSF in a 1:100 dilution, and protease inhibitor in a 1:200 dilution into lysis buffer. o Incubate cells in Lysis buffer on ice for 10 min. o After incubation, centrifuge sample at 10,000 g for 15 mins. • Protein quantification using BCA assay. o Prepare BCA assay solution for all samples in a falcon tube • 200 uL reagent A for each sample, 1:50 dilution of reagent B. o Prepare standards by serially diluting bovine serum albumin to known concentrations. o In a 96 imaging plate, add 200 uL of BCA assay solution and 10 uL of sample or standard. o Incubate at 37 C for 30 min. o Image plate using a plate reader. • Protein separation using Polyacrylamide gel electrophoresis. o Create a 10% Polyacrylamide gel according to protocol. o Mix 6.5 ug protein from each sample with 10 uL Laemmli solution, and place on heating block at 95 C for 5 mins. o Prepare biotinylated ladder by mixing 1 uL of ladder solution with 15 uL of Laemmli solution. o On gel, load 10 uL kaleidoscope ladder, 11 uL biotinylated ladder, and the entire volume for the samples. Run at 60 V until sample enters gel front. Then, run at 75 V for 2 hours. • Protein transfer to membrane. o Make transfer buffer by adding 100 mL 10x transfer buffer, 200 mL methanol, and 700 mL dH2O. o Cut out membrane of proper size. o Charge membrane by washing in methanol for 15 s, then water for 5 min, then soak in transfer buffer. o Assemble transfer apparatus in correct order. From top to bottom: foam pad, Whatman paper, membrane, gel, Whatman paper, foam pad. • When placing gel, ensure that gel is not dry for too long, and that it lines up with membrane. o Place assembled apparatus into transfer buffer and run in ice at 100 V for 1 hour. • Incubation with primary antibodies (PTEN). o Remove membrane from transfer apparatus. o Wash once with water, and 4-5 times with TBST buffer for 5 min each on a rocker. o Prepare a 1:1000 dilution of PTEN antibody in 5% milk as blocking agent. o After washes, add 10 mL of antibody solution to membrane. o Incubate membranes in antibody solution on rocker at 4 C overnight. • Incubation with secondary antibodies (PTEN). o Remove primary antibody solution from membranes. o Wash 4-5 times with TBST buffer on rocker with 10 min for each wash. o Prepare 1:1000 dilution of secondary anti-rabbit antibodies and 1:2000 dilution of anti-biotin antibodies in 1% milk as blocking agent. o After washes, add 10 mL of secondary antibody solution to membranes. o Incubate on rocker at room temperature for 1 hour. • Imaging (PTEN) o Remove secondary antibody solution from membranes. o Wash 5-10 times with TBST buffer on rocker. o Prepare developing solution. • 4.5 mL ddH2O, 250 uL developing buffer 1, and 250 uL developing buffer 2. o After wash, blot membrane to remove excess liquid and lay membrane on flat surface. o Add developing solution on membrane until uniformly covered. o Leave developing solution on membrane for 20 s, then blot off excess liquid. o Lay membrane on cassette, wrap in plastic wrap, and close cassette to protect from light. o In dark room, place film on top of membrane inside cassette, and let film develop for ~30 s. o Remove film, and place in developer to obtain image.

• GAPDH Detection o Follow same procedure for primary antibody incubation and secondary antibody incubation using the anti-GAPDH antibody instead of the anti-PTEN antibody. For imaging, develop film ~3 s instead ~30 s.

Results:

• Total protein concentrations using BCA protein assay.

<figure> <img src="http://openwetware.org/images/3/31/2bcacurve.png"height="306" width="446"/> <figcaption>Figure 1: BCA standard curve using bovine serum albumin dilutions of known concentrations.</figcaption> </figure> The linear trendline for the standard curve fit well with the experimental data, with a coefficient of correlation over 0.99. However, the resolution of the data did not extend to the lowest concentration tested (0.031 ug/uL). <figure> <figcaption>Table 1: Total protein concentrations in ug/uL in different samples calculated using the BCA protein assay.</figcaption> <img src="http://openwetware.org/images/a/ad/3_table1.png"height="160" width="572"/> </figure> The total protein concentrations for the different samples showed high, random variability.

• Western blot analysis

24 hrs

<figure> <img src="http://openwetware.org/images/4/4e/4fig2.png"height="134" width="438"/> <figcaption>Figure 2: Western blot images showing relative expression of PTEN and GAPDH. The top bands are PTEN, and the bottom bands are GAPDH.</figcaption> </figure> The images of the blots were ideal, with specific antibody binding, resulting in clear bands and low background. The 5% milk blocking solution proved to be better than the bovine serum albumin solution, which caused non-specific binding of antibodies, resulting in high levels of uneven background levels. For the PTEN bands, the antibody stained well, with sharp intense bands. The band front was slightly crooked, but was probably the result of loading errors rather than differences in the protein composition. Qualitatively, the Br mir sample displayed the highest concentration of PTEN levels, whereas the cells only sample showed the least. For BO, the scr sample seemed to have higher intensity, suggesting a higher expression of PTEN relative to the mir sample. For the GAPDH bands, the antibody staining was not ideal, since the bands were not sharp and showed variable intensity. The development time was also too high, since the bands bled into each other. In terms of expression, the different samples seemed to show similar levels of band intensity, suggesting that the treatment did not affect GAPDH expression

<figure> <figcaption>Table 2: Densitometric analysis of western blot band intensities to determine PTEN expression relative to housekeeping protein (GAPDH) and different controls.</figcaption> <img src="http://openwetware.org/images/a/a2/5table2.png"height="176" width="458"/> </figure> <figure> <img src="http://openwetware.org/images/c/cb/6fig3.png"height="348" width="496"/> <figcaption>Figure 3: Graph showing relative expressions of PTEN in cells incubated with different structures after 24 hours incubation.</figcaption> </figure>

<figure> <img src="http://openwetware.org/images/0/0d/7fig4.png"height="346" width="494"/> <figcaption>Figure 4: Graph showing relative expressions of PTEN in cells incubated with different structures after 24 hours incubation.</figcaption> </figure> The densitometric analysis confirmed the qualitative assessments made using the blot images. After normalization to the GAPDH band, the highest intensity band was the Br scr, with the lowest intensity being the cells only sample (Table 2). Further normalization to the cells only control showed that all four structure treatments caused a significant increase in the PTEN levels, as shown in Figure 3. However, further normalization to the scrambled overhangs control highlighted the discrepancies between the scrambled and the complementary mir treatments for both structures. For the branch structures, the mir treatment showed an increase in PTEN relative to the scrambled treatment, whereas for Block O the mir treatment showed lower levels of PTEN expression relative to its corresponding scrambled treatment as seen in Figure 4. 48 hrs

<figure> <img src="http://openwetware.org/images/3/33/8fig5.png"height="134" width="438"/> <figcaption>Figure 5: Western blot images showing relative expression of PTEN and GAPDH. The top bands are PTEN, and the bottom bands are GAPDH.</figcaption> </figure>

The characteristics of the blots were at 48 hours were very similar to those at 24 hours, with one significant difference. The sample with the most band intensity was the BO mir sample instead of the Br mir sample. In addition, the BO mir band was visibly more intense than the BO scr sample, while the there was no discernible differences between the intensities of the Br scr and Br mir bands. However, the GAPDH band for the Br scr sample seemed more intense than the that of the Br mir band.

<figure> <figcaption>Table 3: Densitometric analysis of western blot band intensities to determine PTEN expression relative to housekeeping protein (GAPDH) and different controls.</figcaption> <img src="http://openwetware.org/images/d/d3/9table3.png"height="150" width="458"/>

</figure> <figure> <img src="http://openwetware.org/images/0/08/10fig6.png"height="342" width="472"/> <figcaption>Figure 6: Graph showing relative expressions of PTEN in cells incubated with different structures after 48 hours incubation.</figcaption> </figure> <figure> <img src="http://openwetware.org/images/4/48/11fig7.png"height="344" width="494"/> <figcaption>Figure 7: Graph showing relative expressions of PTEN in cells incubated with different structures after 24 hours incubation.</figcaption> </figure> Even though no qualitative conclusion could be drawn about the Branch structures from the blot images, densitometry revealed that the Br mir sample, when normalized to GAPDH, did indeed show a higher PTEN expression relative to the scrambled overhangs expression. Furthermore, in contrast to the 24 hr time point, the BO mir sample also showed a higher level of expression than the BO scr sample. In fact, the increase in expression in the BO sample was greater than that of the Br sample (Figure 7). One continuing trend from the 24 hr time point was the higher level of PTEN expression compared to the cells only sample (Figure 6). 72 hrs:

<figure> <img src="http://openwetware.org/images/9/9e/12fig8.png"height="134" width="438"/> <figcaption>Figure 8: Western blot images showing relative expression of PTEN and GAPDH. The top bands are PTEN, and the bottom bands are GAPDH.</figcaption> </figure> At 72 hours, the blot images were significantly different than the other two time points. Once again, the blots were clear with no background. However, in terms of band intensity, the Br scr PTEN band was significantly more intense than all others, where PTEN bands for Br and BO mir samples were non-existent, suggesting that there was no PTEN present in those cells. The GAPDH bands looked similar to those from the 24 and 48 hr time points. Discussion: The BCA protein assay is conducted to measure the concentrations of total protein in unknown protein extract samples so that equal amount of protein can be used for western blot experiments, thus reducing the number of confounding factors. The assay calculates concentration by comparing the absorbance from the unknown sample to a standard curve generated by samples with known concentrations. The resolution of the assay is therefore equal to the range on the standard curve. The standard curve generated for this experiment had an effective range between 0.125 ug/uL and 2 ug/uL, since the 0.031 ug/uL sample did not fit with the linear trend. However, this was not an issue, since all the unknowns measured had concentrations above 0.125 ug/uL.

The branch and Block O structures with miR-21 complementary overhangs were developed with the intention of sequestering free miR-21 from the cell cytoplasm. Since one of most well studied effects of miR-21 over-expression is the suppression of PTEN protein, it was hypothesized that the level of PTEN expression would increase in samples treated with the mir structures compared to controls such as structures with scrambled overhangs or cells incubated without any structures.

At 24 hours, there was a measurable increase in relative PTEN levels in the branch mir treatment compared to the scr or the cells only treatment, suggesting that the branch treatment was successful in suppressing miR-21 and increasing the levels of PTEN protein expression. At 48 hours, both Branch and Block O mir treatments showed an increase in relative PTEN expressions, further reinforcing the positive outlook for the structures.

However, a few issues persisted. At 24 hours, the PTEN expression in the BO scr control was higher than that of the BO mir treatment, suggesting that the mir structure was unsuccessful in sequestering miR-21 at 24 hours, even though they were successful in doing so at 48 hours. While this may seem contradictory, cellular uptake experiments conducted earlier hinted that the Block O structures were possibly uptaken by cells at a slower rate than the branch structures. If so, it is possible that the Block O structures were not present in cells at the threshold levels necessary to cause a significant decrease in miR-21 at 24 hours, but were present at required levels 48 hours after incubation. In addition, even though the BO mir treatment was unsuccessful at 24 hours, at 48 hours it displayed a higher increase in PTEN than the branch structure. This can possibly be explained by the fact that the Block O structure has more overhangs than the branch structure. If, at 48 hours, the concentrations of Block O and Branch had equilibrated to a similar number, the 12 extra complementary overhangs on the Block O structure may explain the greater effect seen in the Block O sample compared to the branch sample.

Another discrepancy was the increased levels of PTEN in the scrambled control relative to the cells only control. Ideally, neither the structures nor the scrambled overhangs on the structures should have any extraneous effect on the cell, so the cells only and the scrambled overhangs controls should effectively be the same. However, since an increase in PTEN was observed, the structures themselves could be having an unexpected additive effect to the regulation of PTEN. To test this theory, further replicates of the present experiment and experiments with structures without any overhangs must be conducted in the future.

Finally, the result for the 72 hour time point study was unexpected, since the PTEN levels in the mir treatment were completely suppressed, whereas the PTEN levels, especially in the Br scr treatment, were highly elevated. Initially, it was though that this could have been caused by a significant loss of viability in the mir treatments due to the increase in PTEN. However, such an event would also have reduced the amount of housekeeping protein (GAPDH) in the sample, which was not observed. Another possibility may be that the apoptotic mechanism itself reduces the amount of PTEN in cells, and thus shows decreased expression levels at extended time points such as 72 hours. However, further replicated of this experiment and further research into literature must be conducted to draw a conclusion about the causes behind such a phenomenon.

This experiment represents only one replicate, and is therefore not statistically significant. To achieve statistical significance, comparable results must be obtained in at least two or more replicates of the same experiment<A NAME="scroll5"></A>.

</div>

<div id="5"> <h3>5. PTEN mRNA Experiment</h3> Treatments:

<figure> <img src="http://openwetware.org/images/9/9d/1-table.png"height="134" width="438"/> <figcaption>Figure 1: Western blot images showing relative expression of PTEN and GAPDH. The top bands are PTEN, and the bottom bands are GAPDH.</figcaption> </figure>

<figure> <img src="http://openwetware.org/images/b/bc/2table1.png"height="134" width="438"/> <figcaption>Figure 2: Western blot images showing relative expression of PTEN and GAPDH. The top bands are PTEN, and the bottom bands are GAPDH.</figcaption> </figure>

<figure> <img src="http://openwetware.org/images/8/88/3table2.png"height="134" width="438"/> <figcaption>Figure 3: Western blot images showing relative expression of PTEN and GAPDH. The top bands are PTEN, and the bottom bands are GAPDH.</figcaption> </figure>

<figure> <img src="http://openwetware.org/images/2/2d/4table3.png"height="112" width="120"/> <figcaption>Figure 4: Western blot images showing relative expression of PTEN and GAPDH. The top bands are PTEN, and the bottom bands are GAPDH.</figcaption> </figure> <figure> <img src="http://openwetware.org/images/4/4b/5fig1.png"height="224" width="338"/> <figcaption>Figure 5: Western blot images showing relative expression of PTEN and GAPDH. The top bands are PTEN, and the bottom bands are GAPDH.</figcaption> </figure> <figure> <img src="http://openwetware.org/images/8/8a/6table4.png"height="134" width="438"/> <figcaption>Figure 6: Western blot images showing relative expression of PTEN and GAPDH. The top bands are PTEN, and the bottom bands are GAPDH.</figcaption> </figure> <figure> <img src="http://openwetware.org/images/6/6b/7fig2.png"height="134" width="438"/> <figcaption>Figure 7: Western blot images showing relative expression of PTEN and GAPDH. The top bands are PTEN, and the bottom bands are GAPDH.</figcaption> </figure> <figure> <img src="http://openwetware.org/images/d/d4/8fig3.png"height="134" width="438"/> <figcaption>Figure 8: Western blot images showing relative expression of PTEN and GAPDH. The top bands are PTEN, and the bottom bands are GAPDH.</figcaption> </figure> <figure> <img src="http://openwetware.org/images/e/e6/9fig4.png"height="134" width="438"/> <figcaption>Figure 9: Western blot images showing relative expression of PTEN and GAPDH. The top bands are PTEN, and the bottom bands are GAPDH.</figcaption> </figure> <figure> <img src="http://openwetware.org/images/d/d6/10fig5.png"height="134" width="438"/> <figcaption>Figure 10: Western blot images showing relative expression of PTEN and GAPDH. The top bands are PTEN, and the bottom bands are GAPDH.</figcaption> </figure> <figure> <img src="http://openwetware.org/images/a/a3/11table5.png"height="134" width="438"/> <figcaption>Figure 11: Western blot images showing relative expression of PTEN and GAPDH. The top bands are PTEN, and the bottom bands are GAPDH.</figcaption> </figure> <figure> <img src="http://openwetware.org/images/f/f5/12fig6.png"height="134" width="438"/> <figcaption>Figure 12: Western blot images showing relative expression of PTEN and GAPDH. The top bands are PTEN, and the bottom bands are GAPDH.</figcaption> </figure> <figure> <img src="http://openwetware.org/images/7/76/13fig7.png"height="134" width="438"/> <figcaption>Figure 13: Western blot images showing relative expression of PTEN and GAPDH. The top bands are PTEN, and the bottom bands are GAPDH.</figcaption> </figure> <figure> <img src="http://openwetware.org/images/f/fb/14fig8.png"height="134" width="438"/> <figcaption>Figure 14: Western blot images showing relative expression of PTEN and GAPDH. The top bands are PTEN, and the bottom bands are GAPDH.</figcaption> </figure> <figure> <img src="http://openwetware.org/images/e/ef/15fig9.png"height="134" width="438"/> <figcaption>Figure 15: Western blot images showing relative expression of PTEN and GAPDH. The top bands are PTEN, and the bottom bands are GAPDH.</figcaption> </figure> <figure> <img src="http://openwetware.org/images/e/ea/16fig10.png"height="134" width="438"/> <figcaption>Figure 16: Western blot images showing relative expression of PTEN and GAPDH. The top bands are PTEN, and the bottom bands are GAPDH.</figcaption> </figure> <figure> <img src="http://openwetware.org/images/d/d2/17table6.png"height="134" width="438"/> <figcaption>Figure 17: Western blot images showing relative expression of PTEN and GAPDH. The top bands are PTEN, and the bottom bands are GAPDH.</figcaption> </figure> <figure> <img src="http://openwetware.org/images/3/3b/18fig11.png"height="134" width="438"/> <figcaption>Figure 18: Western blot images showing relative expression of PTEN and GAPDH. The top bands are PTEN, and the bottom bands are GAPDH.</figcaption> </figure> <figure> <img src="http://openwetware.org/images/c/c3/19table7.png"height="134" width="438"/> <figcaption>Figure 19: Western blot images showing relative expression of PTEN and GAPDH. The top bands are PTEN, and the bottom bands are GAPDH.</figcaption> </figure>

</div>

<A HREF="#top"><img src="http://openwetware.org/images/4/46/Arrow_up.png" id="fixedbutton"height="40" width="40"></A> <a href="http://openwetware.org/index.php?title=Biomod/2014/OhioMOD/experimentnotes&action=edit">Edit Experiment Notes</a> </body> </div> </html>

<html>

Footer table {

  width: 96.8%;
  max-width:2350px;
  bottom:0px;
  height:50px;   /* Height of the footer */
  font-weight:300;
  background-color: #2E2E2E;
   text-align:center;
   color: white;
   font-size:13px;
   border: 3px white solid;

}

Footer {

   clear: both; /*may be omitted*/
   position: absolute;
   bottom: 12px; 
   background-color:#fffff;
   width: 100%;
   height: 40px; /* or anything you like */
}

</style> </head> <div id="Footer" >

       <table>
           <tr>

<td id="email">Contact us at <a href="mailto:theohiomod@gmail.com">theohiomod@gmail.com</a></td>

           </tr>
       </table>
   </div>

</html>

Biomod/2014/OhioMOD/experimentnotes

Navigation menu

Page actions

Page actions

Personal tools

Navigation

Search

research

Tools