Cells were plated at 2 × 105 cells per well. AccuTarget Predesigned siRNAs specific for human TRPM7, and scramble siRNAs purchased from Bioneer (Daejeon, Korea) were used to knockdown TRPM7 expression. The sequences for human TRPM7 siRNA were as follows: Fwd 5′-GUC UUG CCA UGA AAU ACU CUU-3′ and Rev. 5′GAG UAU UUC AUG GCA AGA CUU-3′ (siRNA #1), and Fwd 5′-AGG AGA AGA UGC AAU UAA ATT-3′ and Rev. 5′-UUU AAU UGC AUC UUC UCC UAG-3′ (siRNA #2). The negative control (NC) group was treated with a non-targeted sequence, and the sequences for NC were as follows: Fwd 5′- UUC UCC GAA CGU GUC ACG UTT-3′ and Rev. 5′-ACG UGA CAC GUU CGG AGA ATT-3′. For transient transfection, Lipofectamine® RNAiMAX (13778–150; Invitrogen, Carlsbad, CA, USA) was used with siRNA (2–200 pmol/μL) to treat the cells for 6 h. Following treatment, the media was changed to a transfection reagent free media containing 10% FBS, and incubated for 48 h at 37 °C in a humidified 5% CO2 atmosphere.
Scramble sirna
Manufactured by Bioneer
Sourced in United States
Scramble siRNA is a laboratory reagent used in RNA interference (RNAi) experiments. It is a non-targeting control siRNA sequence that does not correspond to any known gene. This scrambled siRNA can be utilized to establish baseline cellular responses in RNAi studies, allowing researchers to distinguish specific target gene knockdown effects from non-specific effects.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine rnaimax, by Thermo Fisher Scientific (3 mentions)
Accutarget predesigned sirna, by Bioneer (2 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (1 mentions)
Ex neg m68, by GeneCopoeia (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Hyclone, by GE Healthcare (1 mentions)
Rpmi 1640, by Lonza (1 mentions)
Dulbecco s modified eagle s medium, by Welgene (1 mentions)
G9545, by Merck Group (1 mentions)
Control mirna, by Bioneer (1 mentions)
Apicidin, by Merck Group (1 mentions)
Doxycycline, by Merck Group (1 mentions)
Chloroquine, by Merck Group (1 mentions)
Tubastatin a, by Merck Group (1 mentions)
Anti yap1, by Cell Signaling Technology (1 mentions)
Anti hsp90, by Santa Cruz Biotechnology (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti actin, by Merck Group (1 mentions)
Anti hdac2, by Abcam (1 mentions)
11 protocols using scramble sirna
1
Knockdown of TRPM7 in Human Cells
2
Knockdown and Overexpression of CAD in Cell Lines
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The HeLa S3 cell line, WI-38 cell line, and BC cell lines (5637, RT4, T24, and TCC-SUP) were obtained from the Korean Cell Line Bank (Seoul, South Korea) and American Type Culture Collection (ATCC, Manassas, VA). HeLa S3, WI-38, and 5637 cells were cultured in RPMI 1640 (Lonza, Walkersville, MD) supplemented with 10% fetal bovine serum (FBS, Gibco BRL, Grand Island, NY) and 1% penicillin/streptomycin (HyClone, GE Healthcare, Little Chalfont, UK). RT4 and T24 cells were cultured in Dulbecco's Modified Eagle's Medium (Welgene, Daegu, South Korea), and TCC-SUP cells were cultured in Eagle's Minimal Essential Medium (ATCC). The cells were maintained in a humidified atmosphere of 5% CO2 at 37°C. Accutarget predesigned siRNAs specific for human CAD and scramble siRNAs purchased from Bioneer (Daejeon, South Korea) were used for knockdown of CAD expression. A plasmid expressing CAD transcript variant 2 (#EX-Z0014-M68, designated as ‘pCADv2’) and the blank vector (#EX-NEG-M68) were purchased from GeneCopoeia (Rockville, MD). Transfections were performed using Lipofectamine 3000 or Lipofectamine RNAiMAX transfection reagent, according to the manufacturer's instructions (Invitrogen, Carlsbad, CA).
3
TRPM7 Knockdown Using siRNA
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AccuTarget Predesigned siRNAs specific for human TRPM7 and scramble siRNAs purchased from Bioneer (Daejeon, Korea) were used to knockdown TRPM7 expression. The sequences for human TRPM7 siRNA were Fwd 5′-GUC UUG CCA UGA AAU ACU CUU-3′ and Rev 5′-GAG UAU UUC AUG GCA AGA CUU-3′. For transient transfection, cells were grown to 80% confluence, and Lipofectamine® RNAiMAX (13778-150; Invitrogen, Carlsbad, CA, USA) was used. Knockdown efficiency was determined using Western blot analysis.
4
Alu siRNA Transfection Protocol
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Alu siRNA sequences are 5 -CUUUGGGAGGCCGAGGCGGGCGGAUCA-3 (sense) and 5 -AUCCGCCCGCCUCGGCCUCCCAAAGUG-3 (antisense). Scramble siRNA was from Bioneer. The day before transfection, cells were seeded at 5 × 10 4 cells per well in a 24-well plate in 0.5 ml of DMEM with 10% fetal bovine serum. For each well of transfection, 20 μM of siRNA was diluted into 25 μl Opti-MEM medium and mixed gently. For each transfection, dilute 2 μl of Lipofectamine reagent into 25 μl Opti-MEM and mix. Combined diluted DNA and diluted Lipofectamine reagent, mix gently, and incubate at room temperature for 20 min. While complexes were forming, replaced medium on the cells with 0.45 ml of DMEM growth medium. For each transfection, added complexes onto the cells, and a produced final concentration of Alu siRNA was 150 nM. Incubated cells with the complex at 37 • C in a CO 2 incubator for 2 days. After the incubation time, cells were collected by trypsinization.
5
Murine siRNA Knockdown Protocol
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Murine Hif-1α, Cd36, and Mfg-e8 siRNA were purchased from Dharmacon and scramble siRNA from Bioneer. siRNA sequences were as follows: mouse Hif-1α siRNA, 5’-GGA AAG AGA GUC AUA GAA C-3’; mouse Cd36 siRNA #1 Sense, 5’-CUG AGU AGG UUU UUC UCU U(dTdT)- 3’ and Antisense, 5’-AAG AGA AAA ACC UAC UCA G(dTdT)-3’; mouse Cd36 siRNA #2 Sense, 5’-AGU CAU CAA UGU UCC UAC A(dTdT)-3’, Antisense, 5’-UGU AGG AAC AUU GAU GAC U(dTdT)-3’; mouse Mfg-e8 siRNA #1 Sense. 5’-GAC UGU AUA UGA GGA GCA A(dTdT)-3’, Antisense, 5’-UUG CUC CUC AUA UAC AGU C(dTdT)-3’; mouse Mfg-e8 siRNA #2 Sense, 5’-CAG UAU GUG GAG UCC UAC A(dTdT)-3’, Antisense, 5’-UGU AGG ACU CCA CAU ACU G(dTdT)-3’; scramble siRNA, 5’-AUCCGCGCGAUAGUACGUATT-3’. siRNA transfection was performed with Lipofectamine RNAiMAX (Invitrogen). In brief, 60 pmol of siRNA were mixed with 10 μl of Lipofectamine RNAiMAX in 1ml of pure DMEM medium (Welgene). The mixture was added to BV2 cells that were 70% confluent in 6-well culture dishes without antibiotics. 24 hr after transfection, cells were then resuspended in complete DMEM medium, incubated for another 24 hr, and used for experiments.
6
miR-134-5p Regulation of RUNX2
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miR‐134‐5p mimic, control miRNA, HDAC5 siRNA and scramble siRNA were purchased from Bioneer Corp. Antibodies against RUNX2 (23981, Abcam) and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) (G9545, Sigma) were used at 1:1000 dilution.
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miR‐134‐5p mimic, control miRNA, HDAC5 siRNA and scramble siRNA were purchased from Bioneer Corp. Antibodies against RUNX2 (23981, Abcam) and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) (G9545, Sigma) were used at 1:1000 dilution.
7
Antibody and Reagent Specification Protocol
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Antibodies used were as follows: anti-YAP1, anti-phosphor-YAP1 (S127), and anti-acetyl-lysine were from Cell Signaling Technology (Danvers, MA, USA). Anti-PSME4, anti-histone H3, and anti-HDAC2 were from Abcam (Cambridge, UK). Anti-14-3-3 epsilon was from Thermo Fisher Scientific (Waltham, MA, USA). Anti-HSP90, anti-HDAC6, anti-acetylated-tubulin, and anti-GAPDH were from Santa Cruz Biotechnology (Dalla, CA, USA). Anti-actin, anti-tubulin, anti-HA, and anti-flag were from Sigma (St. Louis, MO, USA). Alexa Fluor 568-conjugated goat anti-mouse IgG was from Molecular Probes (Invitrogen, Waltham, MA, USA).
Reagents used were as follows: apicidin, cycloheximide, chloroquine, actinomycin D, leptomycin B, nicotinamide, tubastatin A, and doxycycline were from Sigma (St. Louis, MO, USA). MG132 was purchased from Cayman (Ann Arbor, MI, USA). Puromycin and blasticidin were from InvivoGen (San Diego, CA, USA).
siRNAs targeting HDAC6, FBXW7, SOCS6, and PSME4 were purchased from Dharmacon (Lafayette, CO, USA). Scramble siRNA was purchased from Bioneer (Daejeon, Korea).
Reagents used were as follows: apicidin, cycloheximide, chloroquine, actinomycin D, leptomycin B, nicotinamide, tubastatin A, and doxycycline were from Sigma (St. Louis, MO, USA). MG132 was purchased from Cayman (Ann Arbor, MI, USA). Puromycin and blasticidin were from InvivoGen (San Diego, CA, USA).
siRNAs targeting HDAC6, FBXW7, SOCS6, and PSME4 were purchased from Dharmacon (Lafayette, CO, USA). Scramble siRNA was purchased from Bioneer (Daejeon, Korea).
8
Sirt1 Silencing in HaCaT Cell Migration
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HaCaT cells seeded in 96-well plates (~60% confluence) were transfected with 100 nM (final concentration) of Sirt1 siRNA or scramble siRNA (Bioneer Corporation) dissolved in serum-free media with an equal volume of a 1.4-1.5% solution of Lipofectamine® (Invitrogen; Thermo Fisher Scientific, Inc.) prepared in serum-free medium. Cells were then incubated for 6 h before washing with PBS and then subjected to the cell migration assay. The independent sequences used for silencing Sirt1 were as follows: Sequence 1, sense, 5′-ACU UUG CUG UAA CCC UGU A(dTdT)-3′ and antisense, 5′-UAC AGG GUU ACA GCA AAG U(dTdT)-3′; sequence 2, sense, 5′-AGA GUU GCC ACC CAC ACC U(dTdT)-3′ and antisense, 5′-AGG UGU GGG UGG CAA CUC U(dTdT)-3′. The sequence for the scramble siRNA was as follows: Sense, 5′-AGA GUU CAA AAG CCC UUC A(dTdT)-3′ and antisense, 5′-UGA AGG GCU UUU GAA CUC U(dTdT)-3′.
9
Silencing Sp1 in MKN1 Cells
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MKN1 cells were seeded on 6-well plates with complete media for 24 h. Then, 20 nM siRNAs against Sp1 (Santa Cruz, Dallas, TX, USA) and scramble siRNA (Bioneer, Daejeon, Korea) were transfected via electroporation (2 pulses, 1100 volt, and 20 msec). Protein and mRNA samples are prepared 72 h after the transfection.
10
Dendritic Cell Differentiation via siRNA Knockdown
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Bone marrow-derived cells (2.5 × 105/well) were seeded into 6-well plates in 2 mL of culture medium and treated with cytokines (rmIL-4 and rmGM-CSF). siRNA transfections were performed using jetPEI reagent according to the instructions of the manufacturer. Cells were transfected with 100 ng/well of CD83 siRNA (Integrated DNA Technologies, CA, USA) and scramble siRNA (Bioneer, Daejeon, Korea) at day 3 after cell seeding. siRNA treatment was three times with 3-day intervals. After the final treatment, 2 hours later, cells were harvested for further analysis.
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