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<h3>Self-assembly </h3> | |||
<li>RNA/DNA hybrid structure</li> | |||
<li>dsRNA structure</li> | |||
<h3>Luciferase-knockdown-assay </h3> | |||
The purpose of the design of oktahedron structure made of dsRNA, was to make a knock-down effect of Renilla luciferase. To investigate the knockdown effect of our structure was transfected into a stable H1299 lung cancer cell line, after which a dual-luciferase assay was performed. | |||
<embed src="knockdown.jpg" width="100%" height="60" align="center"> | |||
<i>Knockdown effect caused by the designed RNA structure studied with dual-luciferase assay. Values are calculated from 3 replicates by transfection with the purified, respectively. uoprensede structure. All control experiments are performed on 4 independent experiments. siEGFP and siEGFP-Cy3 are positive controls with complete base pairing between target and siRNA. siEGFPmis is negative control with no complementary sequence to the EGFP site. "Tranf" are cells exposed only to transfektionsreagens. "Mock" cells are only added RPMI. For each reporter was Renilla luciferase (Rluc) / Firefly luciferase (Fluc) ratio corresponds to "siEGFPmis" control.</i> | |||
The two positive controls gave both a knockdown at around 96% (P = 0.01441 and P = 0.01450), representing significant knock downs. Transfection with the purified structure, a knockdown at around 40% (P = 0.1425), compared to siEGFP mismatch. In cells transfected with a solution containing both the overall structure and tie strings seen a knockdown at around 80% (P = 0.02630) compared to the negative control. The difference in knockdown between the two solutions may be due to the stoichiometric difference in transfection. It was not possible to determine the concentration of the structure of solutions for self-assembly, but the estimated concentration of uoprensede structure transfektionsopløsningen is 16 nM, ie lower than the 40nm as the control solutions contained. The concentration of the purified structure must be lower than 16nM, due to the loss in purification process, which can be give explanation for the lower knock-down effect. The gel analysis confirmed that the structure was present after purification (data not shown). For each knockdown assay, the statistical P-value calculated, respectively. 0.1425 and 0.02630 for the purified and uoprensede structure. Based on these values, there is no significant evidence that the purified structure causes a knockdown, but the uoprensede, however, there is evidence that there is a knock-down effect, if using a P-value for significance at 0.05 . There is therefore a clear basis for further investigation of the knockdown effect. Prior to luciferase assay was performed, cells were examined under light microscope mode. Cells transfected with the purified structure was generally shared well and there were many cells in the wells. Cells transfected with the uoprensede structure had however had it harder, and cell concentration was significantly lower in these wells than in others. This may be due to the many ssRNA staple strands may have started an immune response in cells so that their replication was reduced. The observed luciferase activity in cells transfected with uoprenset structure was therefore also generally lower, giving a larger uncertainty. Since it is the ratio of Firefly luciferase activity and Renilla luciferase activity under investigation data should still be usable. | The two positive controls gave both a knockdown at around 96% (P = 0.01441 and P = 0.01450), representing significant knock downs. Transfection with the purified structure, a knockdown at around 40% (P = 0.1425), compared to siEGFP mismatch. In cells transfected with a solution containing both the overall structure and tie strings seen a knockdown at around 80% (P = 0.02630) compared to the negative control. The difference in knockdown between the two solutions may be due to the stoichiometric difference in transfection. It was not possible to determine the concentration of the structure of solutions for self-assembly, but the estimated concentration of uoprensede structure transfektionsopløsningen is 16 nM, ie lower than the 40nm as the control solutions contained. The concentration of the purified structure must be lower than 16nM, due to the loss in purification process, which can be give explanation for the lower knock-down effect. The gel analysis confirmed that the structure was present after purification (data not shown). For each knockdown assay, the statistical P-value calculated, respectively. 0.1425 and 0.02630 for the purified and uoprensede structure. Based on these values, there is no significant evidence that the purified structure causes a knockdown, but the uoprensede, however, there is evidence that there is a knock-down effect, if using a P-value for significance at 0.05 . There is therefore a clear basis for further investigation of the knockdown effect. Prior to luciferase assay was performed, cells were examined under light microscope mode. Cells transfected with the purified structure was generally shared well and there were many cells in the wells. Cells transfected with the uoprensede structure had however had it harder, and cell concentration was significantly lower in these wells than in others. This may be due to the many ssRNA staple strands may have started an immune response in cells so that their replication was reduced. The observed luciferase activity in cells transfected with uoprenset structure was therefore also generally lower, giving a larger uncertainty. Since it is the ratio of Firefly luciferase activity and Renilla luciferase activity under investigation data should still be usable. | ||
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Revision as of 08:59, 24 July 2011
<html>
<h3>Self-assembly </h3> <li>RNA/DNA hybrid structure</li> <li>dsRNA structure</li>
<h3>Luciferase-knockdown-assay </h3>
The purpose of the design of oktahedron structure made of dsRNA, was to make a knock-down effect of Renilla luciferase. To investigate the knockdown effect of our structure was transfected into a stable H1299 lung cancer cell line, after which a dual-luciferase assay was performed.
<embed src="knockdown.jpg" width="100%" height="60" align="center"> <i>Knockdown effect caused by the designed RNA structure studied with dual-luciferase assay. Values are calculated from 3 replicates by transfection with the purified, respectively. uoprensede structure. All control experiments are performed on 4 independent experiments. siEGFP and siEGFP-Cy3 are positive controls with complete base pairing between target and siRNA. siEGFPmis is negative control with no complementary sequence to the EGFP site. "Tranf" are cells exposed only to transfektionsreagens. "Mock" cells are only added RPMI. For each reporter was Renilla luciferase (Rluc) / Firefly luciferase (Fluc) ratio corresponds to "siEGFPmis" control.</i>
The two positive controls gave both a knockdown at around 96% (P = 0.01441 and P = 0.01450), representing significant knock downs. Transfection with the purified structure, a knockdown at around 40% (P = 0.1425), compared to siEGFP mismatch. In cells transfected with a solution containing both the overall structure and tie strings seen a knockdown at around 80% (P = 0.02630) compared to the negative control. The difference in knockdown between the two solutions may be due to the stoichiometric difference in transfection. It was not possible to determine the concentration of the structure of solutions for self-assembly, but the estimated concentration of uoprensede structure transfektionsopløsningen is 16 nM, ie lower than the 40nm as the control solutions contained. The concentration of the purified structure must be lower than 16nM, due to the loss in purification process, which can be give explanation for the lower knock-down effect. The gel analysis confirmed that the structure was present after purification (data not shown). For each knockdown assay, the statistical P-value calculated, respectively. 0.1425 and 0.02630 for the purified and uoprensede structure. Based on these values, there is no significant evidence that the purified structure causes a knockdown, but the uoprensede, however, there is evidence that there is a knock-down effect, if using a P-value for significance at 0.05 . There is therefore a clear basis for further investigation of the knockdown effect. Prior to luciferase assay was performed, cells were examined under light microscope mode. Cells transfected with the purified structure was generally shared well and there were many cells in the wells. Cells transfected with the uoprensede structure had however had it harder, and cell concentration was significantly lower in these wells than in others. This may be due to the many ssRNA staple strands may have started an immune response in cells so that their replication was reduced. The observed luciferase activity in cells transfected with uoprenset structure was therefore also generally lower, giving a larger uncertainty. Since it is the ratio of Firefly luciferase activity and Renilla luciferase activity under investigation data should still be usable.
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