In the first round of siRNA-mediated knockdown experiments, WM115, WM902B, and A2058 cells were seeded in 200 µl of antibiotic-free growth media in 96-well clear bottom black plates at seeding densities of 3500, 4000, and 1700 cells per well, respectively. After 24 h, cells were transfected using the DharmaFECT 1 transfection reagent (GE Dharmacon T-2001-01) with Dharmacon’s ON-TARGETplus Human SMARTpool siRNAs, including four target-specific siRNAs combined into a single pool to increase the likelihood of effective gene silencing. The SMARTpool siRNAs included KDM4A (L-004292-00-0005), KDM4B (L-004290-00-0005), KDM5A (L-003297-02-0005), KDM5B (L-009899-00-0005), or non-targeting control (D-001810-10-05) and were used at 25 nM. To rule out the possibility of off-targeting by KDM4B siRNAs, we then examined separately the effects of three constituent ON-TARGETplus KDM4B siRNAs (J-004290-08-0005, J-004290-09-0005, J-004290-11-0005) at 25 nM. To evaluate the effect of Braf/Mek inhibition in cells in which either of the proteins were knocked down, WM115 and WM902B cells were further treated with vemurafenib (at 100 nM) plus trametinib (at 10 nM) or DMSO 48 h following siRNA addition. After another 48 h, cells were fixed and analyzed using quantitative immunofluorescence microscopy.
On targetplus human sirna smartpool
Manufactured by Horizon Discovery
Sourced in United Kingdom
ON-TARGETplus human siRNA SMARTpool is a collection of four pre-designed and validated small interfering RNA (siRNA) duplexes targeting a specific human gene. It is designed to provide efficient gene knockdown for use in cell-based assays and research.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine rnaimax, by Thermo Fisher Scientific (6 mentions)
Rnaimax, by Thermo Fisher Scientific (5 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Dharmafect 1, by Horizon Discovery (3 mentions)
Dharmafect 1 transfection reagent, by Horizon Discovery (2 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (2 mentions)
Independent kras sirnas, by Thermo Fisher Scientific (2 mentions)
Hadv b7, by American Type Culture Collection (1 mentions)
Fc 40, by Merck Group (1 mentions)
Usinglipofectamine rnaimax reagent, by Thermo Fisher Scientific (1 mentions)
Rat tail collagen type 1, by Corning (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Tnf α, by Cell Signaling Technology (1 mentions)
Pgl3 basic vector, by Promega (1 mentions)
Dharmafect, by Horizon Discovery (1 mentions)
Attractene, by Qiagen (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
Hiperfect, by Qiagen (1 mentions)
26 protocols using on targetplus human sirna smartpool
1
Dissecting KDM4/5 Epigenetic Regulators
2
Cell Culture and Transfection Techniques
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK293T, COS-7, and HeLa cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and antibiotics. For the generation and maintenance of stable cell lines, neomycin and geneticin were added to the cell culture medium. Transfections of plasmids or siRNA were performed using Lipofectamine 2000 or Lipofectamine RNAiMAX, respectively, following the manufacturer’s instructions (Invitrogen). For experiments in which siRNA and plasmid DNA were both transfected, siRNA transfection was performed first followed by plasmid transfection. ARFGAP1 (L-013321-02), KDELR1 (L-019136-01), RER1 (L-017170-01) ON-TARGETplus Human SMARTpool siRNAs and RER1 (L-017170-11) and customized siRNAs for HRD1 (5′-AAGGUGAUGGGCAAGGUGUUC) and gp78 (5′-AAGACGGAUUCAAGUACCUUU) were purchased from Dharmacon. Scrambled siRNAs were used as negative controls (Invitrogen).
3
GFP-GRs Localization in PRMT-1 Knockdown
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HeLa Cells were transiently transfected with ON-TARGETplus Human SMARTpool siRNA targeting PRMT-1 (Dharmacon #3276) or the scrambled control (10 nM) using the Lipofectamine RNAiMax reagent according to the manufacturer’s instructions (Life Technologies). After 48 h, cells were transfected with the GFP-GRs expressor and, following further 24 h incubation, were fixed and analyzed by confocal microscopy.
4
Silencing HIF1α, HIF2α and VHL in IMR90
5
Silencing Essential Mitochondrial Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gene knock-down was achieved by transfecting HeLa FlpIn TRex cell lines with small double-stranded interfering RNAs (siRNA). Either Dharmacon ON-TARGETplus Human SMARTPool siRNA for HSPA9, PAM16 or individual siRNA for LONP1 were compared with pooled non-targeting control (NTC) or individual NTC siRNA against firefly luciferase GL2. SiRNA was transfected with Lipofectamine RNAiMAX (Thermo Scientific, 13778150), according to manufacturer’s recommendations. Cells were cultured for 96 h after transfection until harvesting. Successful gene silencing was controlled by monitoring protein levels using immunoblots.
6
siRNA Knockdown of HNF4α and CDX2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ON-TARGETplus Human siRNA SMARTpools (HNF4α and CDX2), individual ON-TARGETplus Human individual siRNAs (HNF4α) and ON-TARGETplus Non-targeting siRNA controls (Dharmacon/Thermo Fisher Scientific) were used to transfect cells (2 × 105) at 50 nM in 6-well plate, using Dharmafect 1 transfect reagent, according to the manufacturer instructions. Knockdown efficiency after 72 h RNAi treatment was examined using quantitative RT-PCR and/or Western Blot analysis (Supplementary Fig. 17 ).
7
Adenovirus Infection Protocol for Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human adenoviruses HAdV-C2, HAdV-B7, and HAdV-E4 were purchased from ATCC (catalog numbers VR-846, VR-7 and VR-1572 respectively). A RIDα-null HAdV-C2 mutant virus that deleted 107 base pairs in the amino terminal region of the viral protein was described in [65 (link)]. Adenoviruses were propagated, and multiplicity of infection or MOI was determined by plaque assay in A549 cells, according to standard methods [124 ]. It has been estimated that an MOI of at least 5 to 10 is required to ensure that 100% of cells are infected in tissue culture [125 ]. In preliminary studies, it was established that an MOI of 50 to 100 triggered EGFR stress responses prior to the onset of viral gene transcription, and sufficient RIDα expression to counter these responses, in human A549 cells; and that an MOI of 200 to 250 induced these responses in mouse fibroblasts. The GFP-tagged Vps22 mammalian expression plasmid was a gift from Dr. Cecilia Bucci (Università di Lecce) [126 (link)]. Gene silencing studies were carried out using ON-TARGETplus Human siRNA Smart Pools from Dharmacon (Lafayette, CO) listed in Table 2 , which were introduced to A549 cells using Oligofectamine Reagent exactly as described in [60 (link)].
8
siRNA Transfection Protocol for Cell Culture
9
siRNA-Mediated Knockdown of CRABP2 and FABP5
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RA FLS cells were cultured in six-well plates (14 × 104 cells/well) or in P100 plates (7 × 105 cells/plate) in DMEM medium 10% FBS and 1% glutamine. Cells were transiently transfected with 20 nM of ON-TARGET plus Human siRNA-SMART pools against CRABP2 and against FABP5, or with ON-TARGET plus Non-targeting Pool, as control siRNA, all of them from Dharmacon (GE, UK). Transfections were performed using DharmaFECT 1 (Dharmacon) in Opti-MEM (Life Technologies, Thermo Fisher Scientific, MA, USA). Experiments were performed 72–144 h after siRNA transfection, as indicated. The degree of suppression was determined by real-time quantitative polymerase chain reaction (qPCR) and by Western blotting.
10
Fibroblast-embedded collagen microtissue generation
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!