Hs_TLR3_5 FlexiTube siRNA

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	siRNA concentration-10–20 nm Add diluted siRNA to diluted Lipofectamine® RNAiMAX Reagent (1:1 ratio). Incubate for 15 minutes at room temperature and add to cells. Incubate cells for 1–2 days at 37°C.

Protocol tips
siRNA concentration-10–20 nm Add diluted siRNA to diluted Lipofectamine® RNAiMAX Reagent (1:1 ratio). Incubate for 15 minutes at room temperature and add to cells. Incubate cells for 1–2 days at 37°C.

Publication protocol

Silencing of TLR3 and TRIF
SW620 or HT29 cells were transfected for 24–48 h with 10–20 nm siRNA against TLR3 (TLR3_5, Qiagen, SI02630768; TLR3_8, Qiagen, SI02655156) or non-silencing control siRNA (Qiagen, SI03650325) using Lipofectamine RNAiMAX (Invitrogen) for TLR3 knockdown or with siRNA against TICAM-1/TRIF (Ambion, s45115) for TICAM/TRIF knockdown using Lipofectamine RNAiMAX. siRNA and transfection reagent (ratio, 1:2) were preincubated for 15 min in RPMI medium before being added to newly seeded cells. Knockdown of targets were confirmed in cell lysates by qPCR.

Silencing of IRF3 and IRF7
HT29 cells were transfected for 24 h with 10 nm siRNA against IRF3 (IRF3.4, Qiagen, SI02626526) or IRF7.1 (SI00448672) or non-silencing control siRNA (Qiagen, SI03650325) using Lipofectamine RNAiMAX (Invitrogen). siRNA and transfection reagent (ratio 1:2) were preincubated for 15 min in RPMI before being added to newly seeded cells. Knockdown of targets was confirmed by qPCR.

Silencing of CHC 1 and UNC93B1
CHC and UNC93B1 were silenced in HT29 or SW620 cells by complexing siRNAs (20 nm) with siLentFect (Bio-Rad) (ratio, 1:3) in Opti-MEM (Invitrogen) for 30 min before cells in solution were added to the transfection mixture and plated in 24-well plates. After 26 h of transfection, cells were treated with a new transfection mixture and incubated for an additional 20 h before stimulation experiments. To silence CHC, HT29 cells were transfected with non-silencing RNA (ON-TARGETplus NON-targeting Pool, Dharmacon, D-001810-10-05, 20 nm) or siRNA against CHC 2 (GCAATGAGCTGTTTGAAGA) or CHC 3 (TGACAAAGGTGGATAAATT) (Dharmacon, LQ-004001-00, 20 nm). To determine CHC protein knockdown, HT29 cells were lysed and assayed by Western blotting using an antibody against clathrin heavy chain 1 (X22, ABR Affinity Bioreagents, MA1-065) or α-tubulin (Santa Cruz Biotechnology, sc5286) as an endogenous control. To silence UNC93B1, cells were transfected with negative control siRNA (Qiagen, SI03650325, 20 nm) or a pool of UNC93B1_4 and UNC93B_6 FlexiTube siRNA (SI00756252 and SI04307912, Qiagen) (10 + 10 nm). Knockdown of UNC93B1 was confirmed in cell lysates by qPCR.

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Manufacturer protocol

Download the product protocol from Qiagen for Hs_TLR3_5 FlexiTube siRNA below.

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