Materials

• TransIT X2 (Manual)
  • 在使用前應該回溫到室溫並且gently vortex

注意事項

1. Optimize reaction conditions for each cell type to ensure successful transfections.
2. Cell density (% confluence) at transfection. The recommended cell density for most cell types is ≥ 80% confluence. Determine the optimal cell density for each cell type in order to maximize transfection efficiency.
3. Ratio of TransIT-X2 to DNA. Determine the best TransIT-X2:DNA ratio for each cell type. Start with 3 μl of TransIT-X2 per 1 μg of DNA. Vary the amount of TransIT-X2 from 2–6 μl per 1 μg DNA to find the optimal ratio.
4. Presence of antibiotics. Antibiotics may inhibit transfection complex formation and therefore should be excluded from the complex formation step.
5. Transfection incubation time. The optimal incubation time can be determined empirically by testing a range from 24–72 hours post-transfection, depending on the stability of the target mRNA and its encoded protein.
6. siRNA dilution. Dilute siRNA using the manufacturer’s recommended buffer. Alternatively, use 100 mM NaCl in 50 mM Tris, pH 7.5, made with RNase-free water. Do not use water alone to dilute siRNA, as this may result in denaturation of the siRNA at low concentrations.
7. siRNA concentration. siRNA used for transfection should be highly pure, sterile, and the correct sequence. Depending on the type of experiment, the optimal final siRNA concentration for transfection is typically within the range of 10–50 nM. As a starting point, we recommend 25 nM siRNA (final concentration in well).
8. Do not use DNA prepared using miniprep kits as it might contain high levels of endotoxin.
9. A high or low cell passage number can make cells more sensitive and refractory to transfection. Maintain a similar passage number between experiments to ensure reproducibility.

Optimization SOP for each cell type

Materials

1. Cell line
2. siRNA
3. plasmid expressing GFP or [target gene of siRNA] fused with GFP
4. 12 well plate (表面積~ 1/2.5 of 6 well plate) (目標for 6well plate & 6 cm2 plate)

測試parameters (粗體是base condition)

1. cell density

   1. for 1*10^6 per 6well: 4*10^5 per 12 well
   2. for 2*10^6 per 6well: 8*10^5 per 12 well

2. Transit-X2 to DNA ratio

   1. 1:2.5

   2. 1:6

3. Transfection incubation time

   1. 24hr
   2. 48hr
   3. 72hr

4. siRNA concentration

   1. 25nM
   2. 50nM

Base condition & validation assay

1. 全組別 (非最終使用)
   1. MOCK
   2. GFP
   3. [gene]-GFP
   4. [gene]-GFP+si[gene]
2. for optimal cell density
   1. MOCK
   2. GFP (4*10^5 per 12 well)
   3. GFP (8*10^5 per 12 well)
   4. [gene]-GFP (4*10^5 per 12 well)
   5. [gene]-GFP (8*10^5 per 12 well)

3. for optimal Transit-X2 ratio

   1. MOCK

   2. GFP (1:2.5)

   3. GFP (1:6)

   4. [gene]-GFP (1:2.5)

   5. [gene]-GFP (1:6)

4. for optimal incubation time

   1. MOCK (fix@24hr)

   2. [gene]-GFP (fix@24hr)

   3. [gene]-GFP (fix@48hr)

   4. [gene]-GFP (fix@72hr)

5. for optimal siRNA concentration

   1. MOCK

   2. [gene]-GFP

   3. [gene]-GFP+si[gene] 25nM

   4. [gene]-GFP+si[gene] 50nM

References

1. TransIT-X2® Dynamic Delivery System - Mirus Bio LLC