A siGENOME library (Dharmacon, Lafayette, CO, USA) containing a pool of 4 distinct siRNA duplexes (6.25 pmol) in each well that target a specific candidate gene or in separate wells, positive and negative controls [6 (link),7 (link)], was employed in the scQuantIM screen. Standard reverse transfection with DharmaFECT 2 transfection reagent (Dharmacon, Lafayette, CO, USA) was performed according to manufacturer’s instructions. Cells were seeded, allowed to grow for 4 (HCT116) to 6 days (hTERT) at 37 °C, fixed (4% paraformaldehyde) and counter-stained (Hoechst 33342) to visualize nuclei [7 (link)]. Subsequent direct tests employed ON-TARGETplus siRNA duplexes (Dharmacon, Lafayette, CO, USA) in a pooled format as detailed elsewhere [6 (link)]. Gene silencing was confirmed by Western blot 4 days post-transfection as described [23 (link)]. Membranes were blotted with the primary antibody at the indicated dilutions (Table 1 ) and visualized using secondary antibodies conjugated to horseradish peroxidase. Blots were imaged on a MyECL Imager (Thermo Scientific, Mississauga, ON, Canada) using standard chemiluminescence.
On targetplus sirna duplexes
Manufactured by Horizon Discovery
Sourced in United States
ON-TARGETplus siRNA duplexes are synthetic short interfering RNA (siRNA) molecules designed for gene silencing applications. They are used to target and suppress the expression of specific genes in various cell lines and model systems.
Automatically generated - may contain errors
Lab products found in correlation
6 protocols using on targetplus sirna duplexes
1
Systematic Screening of Candidate Genes
2
Screening DExD/H Box Helicases in KSHV Reactivation
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Vero rKSHV.219 cells were seeded in individual wells of a 48-well plate at 200 μL of 9.5 × 104 cells/mL in DMEM/10% FBS without antibiotics and grown overnight. A Lipofectamine RNAiMAX transfection (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) was performed with a pool of 5 ON-TARGETplus siRNA duplexes that target a particular DExD/H box helicase or scrambled control (Dharmacon, Horizon Discovery, Lafayette, CO, USA) for a final siRNA concentration of 50 nM as per the manufacturer’s instructions. The list of the 22 DExD/H box helicase targets used in the knockdown screen [40 (link)] is presented in Table S1 . The sequences of siRNAs against specific helicases and controls are in Table S2 . After 28 h, the knock-down of control RNAi GFP was confirmed and all siRNA containing media was replaced with 10% FBS media containing 20 ng/mL TPA, and 2 mM NaB to induce lytic reactivation. Fluorescence images were acquired on a Leica DMI 4000 microscope, cell counts were recorded, and DNA was extracted at 48 h post-induction. Three random fields of vision were selected to quantify the adherent Vero cells prior to the 29 h post-induction time point. After the 36 h time point, lytic Vero cells were no longer adherent and could not be quantified.
3
siRNA-Mediated Gene Silencing Protocol
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Cells were transiently transfected with siRNA duplexes using RNAiMax (Invitrogen) as described elsewhere[18 (link)]. ON-TARGETplus siRNA duplexes targeting SMC1A and GAPDH were purchased (Dharmacon) and employed as either individual siRNAs (100 nM total) or as a pool comprised of four unique siRNAs (25 nM each or 100 nM total) targeting distinct regions of the coding sequence. Gene silencing was confirmed by standard Western blots (see below) four days post-transfection.
4
Silencing of SKP2 Protein Expression
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SKP2 silencing was performed using RNAiMAX (Life Technologies, Burlington, ON, Canada) and ON-TARGETplus siRNA duplexes (Dharmacon, Horizon Discovery Biosciences Ltd., Cambridge, UK). Four individual siRNA duplexes targeting distinct coding regions of SKP2 mRNA (siSKP2-1, -2, -3 or -4) or a pooled siRNA (siSKP2-Pool) comprised of equal molar amounts of each individual siRNA were used, along with a non-targeting siRNA (siControl). Silencing efficiencies were determined using semi-quantitative western blot analyses four days post-transfection [53 (link)], using antibodies and dilutions specified in Table S1 . Semi-quantitative image analyses were employed to determine relative protein expression levels in which SKP2 abundance (band intensities) were normalized to the corresponding loading control (Cyclophilin B) and are presented relative to siControl in the siRNA-based experiments or a non-targeting-control (NT-Control) for the CRISPR/Cas9 clone experiments.
5
Investigating USP22 Silencing and H2Bub1 Abundance
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ON-TARGETplus siRNA duplexes (Dharmacon) were employed either as individual siRNA duplexes (siUSP22-1, -2, -3 or -4) targeting distinct coding regions of the targeted mRNA, or as a pool (siUSP22-Pool) comprised of equimolar amounts of the four individual siRNAs. A negative control siRNA (siControl) was employed in all silencing experiments. Cells were transfected using RNAiMAX (ThermoFisher Scientific) according to the manufacturer’s instructions. Gene silencing was confirmed by Western blot as detailed elsewhere [21 (link),98 (link)] using the antibodies and dilutions indicated in Table S10 . Semi-quantitative Western blot analysis was performed with ImageJ software, where the protein of interest was normalized to the respective loading control (cyclophilin B or α-tubulin) and presented relative to siControl (100%; Figures S7 and S8 ). To assess H2Bub1 abundance, acid-based histone extractions were performed as described elsewhere [99 (link)] and the soluble and histone fractions were analyzed by Western blot to assess USP22 silencing efficiency and H2Bub1 abundance, respectively. H2B was employed as loading control for the histone fraction and semi-quantitative Western blot analysis was performed as described above. Figures were assembled in Photoshop CS6 (Adobe).
6
FBXO7 Silencing Protocol for Functional Studies
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FBXO7 silencing was conducted by transfecting ON-TARGETplus siRNA duplexes (GE Dharmacon) into cells using RNAiMAX (Life Technologies). Four individual siRNA duplexes (siFBXO7-1, -2, -3, -4) targeting unique regions within the FBXO7 coding sequence or a pool (siFBXO7-P) comprised of equimolar amounts of each individual siRNAs and a non-targeting control (siControl) were employed. The two most efficient siRNA duplexes (siFBXO7-2 and -4) were initially identified, and along with the siFBXO7-P were employed in all subsequent experiments. Silencing efficiencies were assessed by western blot 4 days post-transfection as detailed previously (67 (link)) with the antibodies and dilutions indicated inSupplementary Material, Table S12 . Relative protein expression levels were determined using semi-quantitative image analysis in which FBXO7 band intensities were first normalized to the corresponding loading control (α-tubulin or cyclophilin B) and are presented relative to siControl (siRNA) or NT-Control (CRISPR/Cas9).
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FBXO7 silencing was conducted by transfecting ON-TARGETplus siRNA duplexes (GE Dharmacon) into cells using RNAiMAX (Life Technologies). Four individual siRNA duplexes (siFBXO7-1, -2, -3, -4) targeting unique regions within the FBXO7 coding sequence or a pool (siFBXO7-P) comprised of equimolar amounts of each individual siRNAs and a non-targeting control (siControl) were employed. The two most efficient siRNA duplexes (siFBXO7-2 and -4) were initially identified, and along with the siFBXO7-P were employed in all subsequent experiments. Silencing efficiencies were assessed by western blot 4 days post-transfection as detailed previously (67 (link)) with the antibodies and dilutions indicated in
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