Team:TecCEM/Demonstrate
Demonstrate
Math modeling with our results
We did a real time PCR, that demonstrates the mRNA expression vs the amounts of siRNA. The theoretical values are demonstrated as T-Xm and the values obtained in the experiment are represented as Xm. The d and θ were adjusted, .005 and 29.1 respectively, the obtained results prove that the transfection efficiency is of 42.9%. The maximum degradation rate observed as d resulted to be even lower than expected, the first value was .001.
The following table shows the values for each point using the equation.
| X | Xm | T-Xm |
|---|---|---|
| 0 | 1 | 1 |
| 20 | 0.9 | 0.97992 |
| 40 | 0.6 | 0.632184 |
| 60 | 0.57 | 0.285714 |
| 100 | 0.25 | 0.177328 |
The following graph shows that the mRNA degradation does exist, but it doesn’t have the same behavior it is not as reduced as the mode, this means that at a bigger concentration the greater the degradation induced by the siRNA.
siRNA confirmation
The siRNA confirmation protocol was made to test, with the real time PCR, the presence of the siRNAs designed by the iGEM Tec Cem team. The four devices capable of producing the siRNAs were tested: BSLA-SOD, BSLA-WNT, BSLA-RACI and BSLA -AWD. The protocol used for this purpose is based on the liquid northern hybridization proposed by Wang X., Tong Y. and Wang S. (2010). The protocol is described as a novel form to detect miRNAs. We expect to detect the siRNAs that our different constructs can produce taking advantage of their hybridization with the forward and reverse templates of their DNA sequences and the ability of the exonuclease to digest the non-hybridized RNA sequences. First BSLA siRNA confirmation
Figure 1. RNA from the HTT115 strain witouth the siRNA construct and with the siRNA construct (BSLA-AWD) with different hybridization oligos and treatments in a 12% no-denaturing gel at 100 Volts..
Figure 1. RNA from the HTT115 strain witouth the siRNA construct and with the siRNA construct (BSLA-AWD) with different hybridization oligos and treatments in a 12% no-denaturing gel at 100 Volts..
Figure 2. RNA from the HTT115 strain with the siRNA construct (BSLA-SOD, BSLA-WNT and BSLA-RACI) with different hybridization oligos and treatments in a 12% no-denaturing gel at 100 Volts.
Second BSLA siRNA confirmation
Figure 3. RNA from the HTT115 strain witouth the siRNA construct and with the siRNA construct (BSLA-AWD) with different hybridization oligos and treatments in a 12% no-denaturing gel at 100 Volts.
Figure 4. RNA from the HTT115 strain with the siRNA construct (BSLA-SOD, BSLA-WNT and BSLA-RACI) with different hybridization oligos and treatments in a 12% no-denaturing gel at 100 Volts.
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