▶ Experimental data
• Positive and Negative Control siRNA
| A) Northern Blotting |
B) qRT-PCR |
Figure 1. Effects of Human GAPDH Positive Control siRNA.
HeLa cells were transfected separately with AccuTarget ™ Human GAPDH Positive Control and Negative Control siRNA using Lipofectamine 2000 (Invitrogen) at a final concentration of 100 nM. Total cellular RNA was isolated from transfected cells 24 hours after transfection and subjected to northern blot and real-time PCR analysis. As can be seen from Fig.1-B, about 3% GAPDH mRNA remained.
• GFP Control siRNA
Figure 2. HeLa cells in a 24-well plate were cotransfected with 200 ng of CMV-GFP plasmid and 10 nM of GFP siRNA using Lipofectamine 2000 transfection reagent. Next day, the expression of GFP was observed by using a Nikon Eclipse TS100 epifluorescence microscope. In contrast to bright green fluorescence of GFP protein in NC-siRNA-transfected cells, no fluorescence was detected from GFP-siRNA-transfected cells, indicating efficient knockdown of GFP by using our positive control GFP-siRNA.
• Luciferase Control siRNA
Figure 3. HeLa cells in a 6-well plate were cotransfected with 400 ng of CMV-luc plasmid and 10 nM of luciferase siRNA using lipofectamine 2000 transfection reagent. Next day, cells were harvested and assayed for luciferase activity. As shown in Fig. 3, cotransfection with our positive control luciferase siRNA led to efficient knockdown of luciferase activity (85% - 95% knockdown compared to luciferase activity of NC-siRNA-transfected cells).
• Mouse Positive Control siRNA
Figure 4. Effects of Mouse Lamin A/C Positive Control siRNA.
Hepa1-6 cells were transfected separately with AccuTarget ™ Mouse Lamin A/C Positive Control and Negative Control siRNA using Lipofectamine RNAimax (Invitrogen) at a final concentration of 1 nM. Total cellular RNA was isolated from transfected cells 24 hours after transfection and subjected to real-time PCR analysis.
Figure 5. Effects of Mouse Ppib (Cyclophilin B) Positive Control siRNA.
Hepa1-6 & NIH 3T3 cells were transfected separately with AccuTarget ™ Mouse Ppib (Cyclophilin B) Positive Control and Negative Control siRNA using Lipofectamine RNAimax (Invitrogen) at a final concentration of 5 & 1 nM. Total cellular RNA was isolated from transfected cells 24 hours after transfection and subjected to real-time PCR analysis.
