Lipofectamine® 2000 Transfection Reagent

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	Do not add antibiotics to the Transfection Medium

Protocol tips
Do not add antibiotics to the Transfection Medium

Publication protocol

SKOV3 cells were seeded in six-well plates (2×105 cells/well), incubated for 24 hours, then transfected with Notch1 siRNA/DMAPA-chems complexes (N/P =80, 100, or 120) at an siRNA dose of 50 nM. In addition, different siRNA doses (30, 50, 80, or 100 nM) of Notch1 siRNA/DMAPA-chems complexes at a fixed N/P ratio of 100 were also investigated. SKOV3 cells without any disposure were used as the control, while SKOV3 cells cultured with the nonspecific siRNA/Lipo complex were grouped as the NC. Notch1 siRNA/lipofectamine 2000 served as a positive control. The cells were then cultured at 37°C for 5 hours and rinsed with PBS solution. The medium was substituted with fresh serum-supplemented medium and further cultured at 37°C. After incubation for 48 hours, Trizol reagent was used to extract total RNA from SKOV3 cells. qRT-PCR was performed as described in the SYBR Premix Ex Tap kit procedure instructions: denaturation, 1 cycle at 95°C for 2 minutes; amplification, 40 cycles at 95°C for 15 seconds, 60°C for 30 seconds; final extension, 70 cycles at 95°C for 1 minute, 60°C for 1 minute, 60°C to 95°C (10 s/cycle, 0.5°C/cycle), using a sequence detection system (AB VIIA 7DX, Thermo Fisher Scientific). A value of 2−ΔΔCt was figured out to estimate Notch1 mRNA expression

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