We obtained siGENOME ON-TARGETplus SMARTpool human GnRH-I receptor siRNA, human EGF siRNA, human Twist siRNA, human N-cadherin siRNA, and siCONTROL NON-TARGETINGpool siRNA from Dharmacon (Lafayette, CO, USA). We transfected the cells with siRNA (100 nM) using Lipofectamine RNAiMAX. After a 24 h transfection, the medium was removed and changed to a fresh serum-free medium. Cells were transfected with 100 nM si-GLO (Dharmacon) for 24 h, and the siRNA transfection efficiency was verified via fluorescent microscopy.
Siglo
Manufactured by Horizon Discovery
Sourced in United States
SiGLO is a specialized laboratory equipment designed for high-throughput cell engineering applications. The core function of SiGLO is to enable efficient genome editing and cell line generation through a streamlined workflow.
Automatically generated - may contain errors
Lab products found in correlation
Sicontrol non targeting pool sirna, by Horizon Discovery (3 mentions)
Sicontrol, by Horizon Discovery (2 mentions)
Opti mem 1, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Dharmafect 1, by Horizon Discovery (1 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (1 mentions)
On targetplus non targeting control sirna, by Horizon Discovery (1 mentions)
Mircury lna mirna inhibitor, by Qiagen (1 mentions)
On targetplus smartpool, by Horizon Discovery (1 mentions)
Hiperfect reagent, by Qiagen (1 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (1 mentions)
Celltrace violet dye, by BD (1 mentions)
Lipopolysaccharides (lps), by Thermo Fisher Scientific (1 mentions)
Anti cd40, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Interleukin 4 (il 4), by R&D Systems (1 mentions)
Epi 2500 electroporator, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse alexa 488, by Thermo Fisher Scientific (1 mentions)
Axio imager 2, by Zeiss (1 mentions)
Primaria, by BD (1 mentions)
18 protocols using siglo
1
Optimizing siRNA Transfection Efficiency
2
MDA5 and SETDB1 Regulate HSC Function
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Lineage-negative cells from WT or Mda5−/− mice that were cultured as described above were transfected with DharmaFECT1 (T-2001-02, Dharmacon) with 50 nM control non-targeting or Mda5 or Setdb1 siRNA (D-001810-10-05, L-065328-00-0005, L-040815-01-0005, Dharmacon) together with siGLO (D-001630-01-05). After 48 h, cells were collected and stained for LSK/SLAM for 30 min at 4 °C in the dark. HSCs were fixed for cell cycle and sorted HSCs were used for CFU-C assays and RT–qPCR. In the case of Setdb1 knockdown, HSCs (LSK/SLAM) were sorted and processed for RNA-seq or cell cycle analysis.
3
Investigating the Role of UCA1 in KSHV-Infected Cells
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wtKSHV-infected or uninfected TIVE cells were plated in 96-well plates (20,000 cells/well for MTS assay) and 48-well plates (250,000 cells/well for wound healing assay). Uninfected and wtKSHV-infected iSLK cells were plated in 96-well plates (8000 cells/well for MTS assay). siRNAs (5nM or 10 nM) against UCA1 (Qiagen) were transfected using Lipofectamine RNAiMAX reagent (ThermoFisher) according to the manufacturer’s protocol. ON-TARGETplus Non-targeting Control siRNA (Dharmacon) was used as the scrambled negative control. At 4 h post-transfection, the serum free medium was replaced by complete Medium-199 (TIVE) or DMEM (iSLK). Comparable transfection efficiencies were ensured by co-transfection of siGLO (Dharmacon).
4
Foxm1 and miRNA Knockdown in NSCs
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Small-interfering-RNA knockdown of Foxm1 was performed in NSCs with ON-TARGETplus SMARTpool (L-057933-01-0005), control ON-TARGETplus Non-targeting siRNA knockdown (D-001810-02-05) (Dharmacon). Hiperfect reagent (Qiagen) was used for transfections. Cells were harvested for assay 72 h post-transfection. Transfection efficiency was > 80%, monitored using the fluorescent control SiGLO (Dharmacon) and calculated as the percentage of fluorescent cells. All experiments were performed using the using the best knockdown efficiency without off-target effects, controlled using three different housekeeping genes (ß−2-microglobulin, Hprt and Gusb) for RNA studies and using Actin or Hsp70 for immunoblots.
Knockdown of miRNA expression was done with miRCURY LNA™ miRNA inhibitor (Exiqon, miR-130b-5p: code 4101000-011; miR-301a-5p: code 4101560-011, miR-15b-3p: code 4101249-011, miR-19a-3p: code 4101300-101), individually or in combination, and a scrambled control, referred to as LNA ctrl (Exiqon mirCURY knockdown probe control A: code 199002-08) (Exiqon). Hiperfect reagent (Qiagen, Hilden, Germany) was used to transfect the siRNA constructs into NSCs. The final concentrations were 10 nM (for single transfection) and 50 nM (for combination transfections). miRCURY LNA™ miRNA inhibitors are conjugated with fluorescent labels for monitoring of transfection efficiency and efficiency rate was 90%.
Knockdown of miRNA expression was done with miRCURY LNA™ miRNA inhibitor (Exiqon, miR-130b-5p: code 4101000-011; miR-301a-5p: code 4101560-011, miR-15b-3p: code 4101249-011, miR-19a-3p: code 4101300-101), individually or in combination, and a scrambled control, referred to as LNA ctrl (Exiqon mirCURY knockdown probe control A: code 199002-08) (Exiqon). Hiperfect reagent (Qiagen, Hilden, Germany) was used to transfect the siRNA constructs into NSCs. The final concentrations were 10 nM (for single transfection) and 50 nM (for combination transfections). miRCURY LNA™ miRNA inhibitors are conjugated with fluorescent labels for monitoring of transfection efficiency and efficiency rate was 90%.
5
siRNA Knockdown in Primary Human PBMCs
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siRNA transfection into primary human PBMCs was accomplished using the Amaxa Nucleofection System and the Human Monocyte Kit according to the manufacturer’s recommendations. ON-TARGET plus SMARTpool; siIL27 siRNA constructs were used at 100 pmol per transfection. Transfection efficiency of siRNA into primary monocytes was assessed using siGLO, a fluorescently labeled control oligo (Dharmacon, Lafayette, CO). Cells were allowed to rest for 18h following transfection before stimulation. At the time of stimulation, non-adherent cells were removed and fresh media was added.
6
Plasma Cell Differentiation and Class Switch Recombination
7
Efficient TRPM2 siRNA Transfection
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Transfection with siRNA was performed as described [31 (link)]. In brief, cells were cultured at a low density to ensure log phase growth. For transfection, 2 × 106 cells were resuspended in 200 μL RPMI 1640 without phenol red. Shortly before transfection, TRPM2 or nontargeting siRNA was added at a concentration of 1 μM. TRPM2 ON-TARGET SMARTpool and the siCONTROL NON-TARGETING pool siRNA were purchased from Dharmacon (Chicago, IL, USA). Cells were electroporated in a 4 mm cuvette in an EPI2500 electroporator (Fischer, Heidelberg, Germany) at 370 V for 10 ms. Immediately after transfection, cells were resuspended in 6 mL prewarmed medium and continued to be cultured as described above. Transfection efficiency as well as viability was determined by transfecting the cells with 400 nM green fluorescence siRNA (siGLO from Dharmacon, Chicago, IL, USA) followed by propidium iodide exclusion dye and flow cytometric analysis.
8
Visualizing HEK 293T Cell Exosomes
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Fifty thousand HEK 293T cells were seeded on DPL‐coated cover slips (Millipore Sigma, P7280‐5MG). The cells were transfected with siGLO (Dharmacon, D‐001610‐01‐05) at a 10 nM concentration in 1 ml of media using RNAiMAX, INTERFERin or JETPRIME as recommended for a well of a 12 well plate. Media was changed 24 h after transfection. Cells were fixed 6 and 48 h after transfection with 4% PFA in PBS for 10 min at room temperature. Cover slips were washed with 1X PBS thrice then stored at 4°C. Cover slips were washed twice with 1X PBS then incubated with 300 μl purified mouse antihuman CD63 antibody (1:100) (BD Pharmingen, 556019) in 1% BSA 0.25% Triton X‐100 in 1X PBS overnight at 4°C in the dark. The following day, the cover slips were washed twice with 1X PBS then incubated with 300 μl goat anti‐mouse Alexa 488 (1:500, Thermo Fisher Scientific, A11029) in 1% BSA 0.25% Triton X‐100 in 1X PBS for 1 h at 4°C. Then, cover slips were washed twice with 1X PBS and mounted with VECTASHEILD solution (Vector Laboratories Inc, H‐1200) and sealed. Images were acquired using a ZEISS Axio Imager 2 equipped with a 63X Plan‐Apochromat 1.4 Oil lens, an AxioCam mRC detector and the ZEN 2.3 analysis software. Filter sets utilised include Blue (Excitation 390/22 nm, Emission 460/50 nm), Green (Excitation 470/20 nm, Emission 517/25 nm), and Red (Excitation 560/40 nm, Emission 690/50 nm).
9
Knockdown of JNK and PARP Using siRNA
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Knockdown of JNK and PARP was performed with 100 nM of targeting siRNA duplexes (catalog no. 6232 and 6304, respectively; Cell Signaling, Inc., Danvers, MA) delivered as lipid-siRNA complexes using Lipofectamine 2000 and Opti-MEM I (Invitrogen) according to the manufacturer’s instructions. In parallel, siControl and siGLO (catalog no. D-001810-10 and D-001630-02; Dharmacon) at a concentration of 100 nM were similarly complexed. By using a reverse transfection method, complexes were added to trypsinized cells within each well in complete tissue culture medium for 24 h, at which point medium was replaced with fresh medium. Western blot analyses verified knockdown compared to actin using the antibodies described above and untreated cells with typical efficiencies of decrease of >95%. Cells were infected 72 h after treatment.
10
Fluorescent siRNA Transfection Assay
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PC-3 (7 × 104 cells/well) and DU145 (7.5 × 104 cells/well) cells were seeded into 24-well plates in 1 mL media and cultured until they reached ~ 70 % confluency prior to treatment. Cells were transfected with 50 nM green fluorescent dye (6-FAM)-labelled siRNA (siGLO; #D-001630-01-05, Dharmacon) in 500 µL media. After 48 h treatment, the nuclei of cells were stained with Hoescht (1 mg/mL) at 1:2000 (v/v) in a fresh antibiotic-free medium for 1–2 h at 37 °C. Cells were then washed with PBS and images were taken using Lionheart Fx instrument (BioTek, USA).
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