The appropriate recombinant SW48 cells were plated in a 10 cm plate in DMEM supplemented with 10% FBS 24 h before transfection. The following day, cells were transfected with siRNAs against NF1 (2 μg) or control siRNA (2 μg) using Lipofectamine 2000. For EGFR knockdown, cells were plated 96 well format in 100 μl of OptiMEM (10%FBS) with 0.1μg of siRNA mixed with 0.5 μl Lipofectamine 2000 per well. 24hours following EGFR siRNA delivery, cells were treated with Cetuximab for 48 hours, and proliferation was measured by MTT assay. Silencer Select Small interfering RNAs were purchased from Thermo Fisher. Silencer Select-NF1 (s56534) was comprised of pooled RNAs targeting exons 2, 10, 16, 18 and 19 in the NF1 mRNA. Silencer Select-EGFR (s565) was comprised of pooled siRNA targeting five unique sequences within exon 2 of the EGFR mRNA. Silencer Select Control siRNA (4390843) was used as negative control. All siRNAs were reconstituted in RNase-free molecular grade water upon arrival from vendor at concentration of 5mM.
Silencer select small interfering rna
Manufactured by Thermo Fisher Scientific
Sourced in United States
Silencer Select small interfering RNA (siRNA) is a laboratory tool designed to facilitate targeted gene silencing. Its core function is to induce post-transcriptional gene knockdown by binding to and degrading specific mRNA molecules, thereby reducing the expression of the target gene.
Automatically generated - may contain errors
5 protocols using silencer select small interfering rna
1
Silencing NF1 and EGFR in SW48 Cells
2
Targeted PARP1 silencing in HS766T cells
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HS766T cells were plated at 80% confluency in six-well plates 1 day before transfection. Silencer Select small interfering RNAs (siRNAs) from Thermo Fisher Scientific were used as follows: negative control no. 1 siRNA (catalog no. 4390843) and PARP1 siRNA (catalog no. 4390824). Transfections were performed using 10 nM siRNA with Lipofectamine RNAiMAX (Thermo Fisher Scientific, catalog no. 13778150).
3
Silencing Heme Stress Pathways
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Silencer select small interfering RNA specific to heme oxygenase-1, heme oxygenase-2, ferritin heavy chain, biliverdin reductase A and negative control siRNA were obtained from Ambion (Thermo Fisher Scientific, Waltham, Massachusetts, US). HAoEC transfection with siRNA was achieved using the Oligofectamine according to the manufacturer’s guide using 10 nmol/L siRNAs. (Thermo Fisher Scientific, Waltham, Massachusetts, US). Cells were then challenged with heme and the expression of ER stress markers was analyzed as described above.
4
siRNA Knockdown of Oncogenic Targets
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Silencer Select small interfering RNA (siRNA) for knock-down of YAP1 (siYAP1 ‘s20366’), ERα (siERα ‘s4823’), ERβ (siERβ ‘s4826’) and DNMT3B (siDNMT3B ‘s4221’) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). These Silencer Select siRNAs are pre-designed and pre-validated for 100-fold potency and 90% less off-target effects. The reverse transfection method was used to perform high-throughput transfection. Lipofectamine 2000 and Opti-MEM Reduced Serum Medium (Life Technologies) were used to transfect siYAP1 at a final concentration of 100 nM into a breast cancer cell line. Silencer Select Negative Control siRNA (Thermo Fisher, catalog number: 4,390,843) was used as a negative control. Approximately 48 h after transfection, cells were used to analyze tumorigenic changes.
5
ATG5 Silencing Enhances Neferine-TRAIL Efficacy
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DU145 cells were seeded 1×105 cells/well in 24-well plates for 24 h and then transfected with Silencer Select small interfering RNA (ATG5 siRNA; oligo ID HSS114103; Sequence GGU UUG GAC GAA UUC CAA CUU GUU U; Invitrogen; Thermo Fisher Scientific, Inc.) using Lipofectamine® 2000 (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Concomitantly, non-targeting siRNA was transfected as a negative control. The cells were incubated with ATG5 siRNA or negative control siRNA for 6 h and the medium was then changed to RPMI-1640 with 10% FBS for 24 h. The cells were then treated with neferine, or neferine in combination with TRAIL.
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