RAW264.7 cells were transfected with miR‐18a mimics, mimics NC, miR‐18a inhibitor or inhibitor NC (GenePharma) with Lipofectamine 2000 (Invitrogen). FAM‐siRNA (GenePharma) was used to detect transfection efficiency. Following transfection, cells were then infected with Mtb (MOI = 10). Cells were lysed with 0.5% TritonX‐100 for 20 minutes at 24 hours after infection, and resulted lysate was cultured on LJ medium for colony‐forming unit (CFU) counting. Meantime, cells were seeded on cover slides in 24‐well plates for acid‐fast staining.
Fam sirna
Manufactured by GenePharma
Sourced in China
FAM-siRNA is a fluorescently labeled small interfering RNA (siRNA) molecule designed for use in gene silencing experiments. The FAM (fluorescein amidite) label allows for the visualization and tracking of the siRNA within cells or tissues.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Anti tgf β, by Abcam (1 mentions)
α sma, by Abcam (1 mentions)
Branched pei, by Polysciences (1 mentions)
Scrambled sirna siscr, by GenePharma (1 mentions)
Lysotracker red, by Thermo Fisher Scientific (1 mentions)
Sdf 1, by Thermo Fisher Scientific (1 mentions)
Anti col 3, by Abcam (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
β actin, by Abcam (1 mentions)
Anti pcna, by Abcam (1 mentions)
Xfect transfection reagent, by Takara Bio (1 mentions)
Axio observer 7, by Zeiss (1 mentions)
Non silencing sirna, by GenePharma (1 mentions)
Hiperfect transfection reagent, by Qiagen (1 mentions)
Melanin, by Merck Group (1 mentions)
Luciferase assay kit, by Promega (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
17 protocols using fam sirna
1
Quantifying Mycobacterial Infection in RAW264.7 Cells
2
Cyclam-Branched PEI Nanoparticles for TGFβ siRNA Delivery
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Cyclam was bought from Vesina Industrial company (Tianjin, China). Branched PEI (MW = 10 kDa) was purchased from Polysciences (Warrington, PA, USA). AMD3100 was obtained from Biochempartner (Shanghai, China). TGFβ siRNA (5-GCAACAACGCCAUCUAUGATT-3), FAM siRNA (5-UUCUCCGAACGUGUCACGUTT-3), scrambled siRNA (siScr) (5-UUCUCCGAACGUGUCACGUTT-3), siRNA targeting luciferase (5-GGACGAGGACGA GCACUUCUU-3) were acquired from Gene Pharma (Shanghai, China). Phosphate buffered saline (PBS) and the assay kits to measure aspartate aminotransferase (AST), alanine aminotransferase (ALT), bicinchoninic acid (BCA), and hydroxyproline (HYP) were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). CCl4 was from Sinopharm chemical reagent (Shanghai, China). Olive oil was bought from Agric-bemvig (Barcelona, Spain). FBS, trypsin, penicillin/streptomycin (Pen-Strep), and RPMI-1640 medium were purchased from Hyclone (Waltham, MA, USA). DMEM and LysoTracker™ Red were obtained from Gibco (CA, USA) and Beyotime (Shanghai, China). G418 and SDF-1 were bought from Life Technologies (Carlsbad, CA, USA). α-SMA, anti-TGFβ, anti-PCNA, anti-Col-III, and β-actin antibodies were purchased from Abcam (Cambridge, MA, USA). 3,3-Diaminobenzidine (DAB) kit was obtained from Boster biological (Hubei, China).
3
Encapsulation and Serum Stability of siRNA
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FAM-siRNA was synthesized by Shanghai Genepharma Co., Ltd and chemically modified by 2'-O-methylation to reduce off-target effects and enhance its stability. Encapsulating siRNAs in PSP complexes was performed as previously reported 20 (link). Briefly, 60 nM PSP and 300 nM FAM-siRNA were subjected to shaking at 1000 rpm for 3h at room temperature. Subsequently, gel shift and serum stability assays were performed for visualizing the estimated siRNA load capacity of PSP and serum stability of PSPS. For estimation of siRNA load capacity, PSPS (molar ratio: 1:20) complexes were subjected to 0.6% agarose gel electrophoresis followed by ethidium bromide staining. Following electrophoretic separation, the agarose gel was exposed to UV transillumination followed by imaging. The unbound siRNA duplex was of 20 bp size, and PSPS was immobile because of its large size. For serum stability of PSPS, 90 μL PSPS was incubated with 10 μL FBS or SFM for 1-24 h at 37°C. Subsequently, 6x gel loading dye was added and the samples were applied to 0.6% agarose gel, and fluorescence spectrophotometer was used to measure the FAM-siRNA fluorescence intensity.
4
Transient Transfection Efficiency in SMI Cells
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The SMI cells from passage 53 were selected to test its transient transfection efficiency for plasmid and siRNA. The SMI cells were seeded in a 6-well plate (at a density of 5 × 105 cells/well). About 60% confluent monolayers were transfected with 5 μg pEGFP-N1 plasmid or 100 pmol FAM-siRNA (GenePharma, Shanghai, China) using Xfect™ Transfection Reagent accordingly manually (Takara, Kusatsu, Japan). The proportion of fluorescence-positive cells was recorded under ZEISS Axio Observer 7 fluorescence microscope (ZEISS) after 48-h transfection.
5
Galectin-3 Knockdown in Eca109 Cells
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The siRNAs (galectin-3 siRNA and non-silencing siRNA) were designed and synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). The siRNA-Gal-3 sequences were as follows: Gal-3-homo-422 (siGal3-1), 5′-GCCACUGAUUGUGCCUUAUTT-3′; Gal-3-homo-746 (siGal3-2), 5′-GUACAAUCAUCGGGUUAAATT-3′. As the non-silencing siRNA (siRNA-control), a scrambled sequence (5′-UUCUUCGAACGUGUCACGUTT-3′), which does not target any known mammalian gene, was used as a negative control. The transfection efficiency of siRNA was evaluated by using negative control FAM-siRNA (Shanghai GenePharma Co., Ltd.), which emitted faint green fluorescence. The lyophilized siRNAs were dissolved in diethylpyrocarbonate-treated water according to the manufacturer's instructions. Transfection of Eca109 cells with galectin-3 siRNA was performed using HiperFect transfection reagent (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Eca109 cells (10×104 cells in 2 ml complete culture medium) were seeded into a six-well plate at 24 h prior to transfection. The cells treated with siRNA were harvested within 48–72 h after transfection for further experiments.
6
Melanin-Based Cellular Assays and siRNA Experiments
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Melanin was obtained from Sigma-Aldrich (St Louis, MO, USA). PLL and bicinchoninic acid assay (BCA) kits were bought from Solarbio (Beijing, People’s Republic of China). Enzyme-linked immunosorbent assay (ELISA) kit was purchased from Cloud-Clone Corporation (Wuhan, People’s Republic of China). Calcein acetoxymethyl ester and propidium iodide (calcein-AM/PI) Double Stain Kit was purchased from YEASEN (Shanghai Yeasen Biotechnology Co. Ltd., Shanghai, People’s Republic of China). The Cell Counting Kit-8 (CCK8) was supplied by DOJINDO Molecular Technologies, Inc. (Shanghai, People’s Republic of China), and Luciferase assay kit was from Promega Corporation (Fitchburg, WI, USA). All products of siRNA including FAM-siRNA and siRNA of nonsense sequences (written as siRNAN.C. for short) were provided by GenePharma Company (Shanghai, People’s Republic of China). The surviving-targeted siRNA (siRNASur: 5′-GCAUUCGUCCGGUUGCGCUTT-3′) and luciferase-targeted siRNA (siRNALuc: 5′-CUUACGCUG AGUACUUCGATT-3′) were also synthesized by GenePharma Company (Shanghai, People’s Republic of China).
7
Silencing OLIG2 in Oligodendrocytes
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Sequences of interfering siRNA segments were as follows: 5-GCCUUCACCUAUGCAAUUATT-3. Non-targeting siRNA with scramble served as a negative control (NC).
OPCs were seeded at a density of 5 × 105 cells per well in six-well plates. After 24 h, the CM was collected. Twenty micromolars FAM-siRNA (8 µl, Genepharma, Shanghai, China) and 3.75 µl Lipofectamine® 3000 (Invitrogen) were each diluted by 125 µl Opti-MEM (Gibco). Diluted siRNA and Lipofectamine® 3000 were mixed and added into the 1750 µl culture medium of OPCs. After 24 h of interference, we changed new culture medium and after 48 h of interference, the CM and cell proteins of three groups (Blank, NC and siRNA) were collected.
OPCs were seeded at a density of 5 × 105 cells per well in six-well plates. After 24 h, the CM was collected. Twenty micromolars FAM-siRNA (8 µl, Genepharma, Shanghai, China) and 3.75 µl Lipofectamine® 3000 (Invitrogen) were each diluted by 125 µl Opti-MEM (Gibco). Diluted siRNA and Lipofectamine® 3000 were mixed and added into the 1750 µl culture medium of OPCs. After 24 h of interference, we changed new culture medium and after 48 h of interference, the CM and cell proteins of three groups (Blank, NC and siRNA) were collected.
8
Synthesis and Purification of c(RGD)2-9R Peptide
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The peptide of cyclic (RGD)2 conjugated with 9R (c(RGD)2-9R) was synthesized and purified at China Peptides Co., Ltd., Shanghai, China. hTERT-targeted siRNA, the stable negative siRNA, and FAM-siRNA for in vitro studies were synthesized by GenePharma Corp., Shanghai, China. All other chemicals were reagent grade and used without purification.
9
Stable siRNA Formulation for In Vivo Delivery
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HER2-siRNA (HER2si) and EGFR-siRNA (EGFRsi) oligonucleotide sequences are provided in Supplementary Table 1 . All siRNAs used for in vivo experiments were modified with 2-O-methylation at all U and C residues of the sense strand to enhance stability, and reduce off-target and immunostimulatory effects. FAM-siRNA (FAMsi) was synthesized by modifying the 5′-end of the sense strand with fluorescein (GenePharma, Shanghai, China). Control groups were transfected with siRNA using Lipofectamine2000 (Invitrogen, Life Technologies, USA) according to the manufacturer's instructions.
10
PEI-Alg NPs for siRNA Delivery Efficiency
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To evaluate siRNA loading efficiency, gel electrophoresis was performed. The different amounts of PEI–Alg NPs were mixed with 1 µL of FAM–siRNA (sense: 5′-UUCUCCGAACGUGUCACGUTT-3′; antisense: 5′-ACGUGACACGUUCGGAGAATT-3′) (GenePharma, Shanghai, China) and incubated for 30 min at room temperature to generate PEI–Alg/FAM–siRNA at different nitrogen/phosphorus (N/P) ratios. All the mixtures were loaded in the wells of 1% (w/v) agarose gel (Biowest, Shanghai, China) and electrophoresed at 100 V for 20 min, stained with ethidium bromide (EB) and visualized on a FR-980A Gel Documentation System (Furi Technology, Shanghai, China). For evaluating efficiency of delivering siRNA with PEI–Alg NPs, transfection of PEI–Alg/FAM–siRNA to LEPCs was examined with a fluorescence microscope (Olympus Corporation, Tokyo, Japan).
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