KIF2A gene silencing was studied to exclude the possibility of an off-target effect by KIF2A-siRNA. The two target sequences of the synthetic oligonucleotides for siRNA 5’-GGCAAAGAGAUUGACCUGG-3’(siRNA-1#) and 5’-CCCUCCUUCAAGAGAUAAUTT-3’ (siRNA-2#) (Ambion, Austin, TX, USA) had similar effects (Additional file 2 : Figure S2), hence the first sequence (50 nM) was employed in our experiments. Nonsense-siRNA (Dharmacon, Lafayette, Colorado, USA) and mock treatment were used as negative controls. MDA-MB-231 cells, upon reaching 2 × 105 cells per well in six-well plates, were transfected with KIF2A–siRNA and Nonsense-siRNA using Lipofectamine 2000 (Invitrogen, Carlsbad, California, USA) in Opti-MEM (Invitrogen). Cells in the mock group were only treated with medium. After 48 hours of treatment, the cells were harvested for gene expression (RT-PCR method see above) and protein expression analysis (Western Blotting method see cancer tissue protein analysis).
Sirna1
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
SiRNA1 is a small interfering RNA (siRNA) product designed for gene silencing applications in a laboratory setting. It functions by targeting and degrading specific mRNA molecules, thereby reducing the expression of the corresponding genes. The core function of SiRNA1 is to facilitate RNA interference (RNAi) for research purposes without further interpretation or extrapolation on its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Sirna2, by Thermo Fisher Scientific (21 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (9 mentions)
Sirna, by Thermo Fisher Scientific (7 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (6 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (6 mentions)
Opti mem, by Thermo Fisher Scientific (5 mentions)
Sirna3, by Thermo Fisher Scientific (4 mentions)
Sirna1, by GenePharma (3 mentions)
Sirna2, by GenePharma (3 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (3 mentions)
Oligofectamine, by Thermo Fisher Scientific (2 mentions)
Scrambled sirna, by Thermo Fisher Scientific (2 mentions)
Scramble sirna, by Thermo Fisher Scientific (2 mentions)
Lipofectamine, by Thermo Fisher Scientific (2 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (2 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (2 mentions)
Nonsense sirna, by Horizon Discovery (1 mentions)
Gene pulser xcelltm, by Bio-Rad (1 mentions)
Neon transfection system, by Thermo Fisher Scientific (1 mentions)
Neon tip with neon transfection system, by Thermo Fisher Scientific (1 mentions)
34 protocols using sirna1
1
Investigating KIF2A Gene Silencing
2
siRNA Knockdown of TBK1 in Cells
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siRNA specifically targeting TBK1 as well as a non-targeting control siRNA was purchased from Santa Cruz biotechnology (siRNA#1) or Ambion (siRNA#2). siRNAs were transfected using Oligofectamine (Invitrogen Corporation) according to manufacturer's protocols.
All data were graphically represented and statistically analysed using Microsoft Office Excel 2003 (Microsoft Corporation, Redmond, WA). In all analyses, means and 95% confidence intervals were estimated. Statistical analysis was performed using Student t-test and values were considered significant when the P value was <0.05.
All data were graphically represented and statistically analysed using Microsoft Office Excel 2003 (Microsoft Corporation, Redmond, WA). In all analyses, means and 95% confidence intervals were estimated. Statistical analysis was performed using Student t-test and values were considered significant when the P value was <0.05.
3
siRNA-Mediated SPINK2 Knockdown
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For SPINK2 knockdown, predesigned siRNAs were purchased from Ambion (siRNA#1: ID_s13362, siRNA#2: ID_s224675). Negative control siRNAs were also obtained from Ambion (Cat #AM4611). Approximately 5 × 106 cells in RPMI1640 medium were transfected with 500 nM siRNAs using electroporation (Bio-Rad Gene Pulser XcellTM) with 0.4 cm cuvettes and the following conditions: voltage, 300 V; and capacitance, 700 µF. Forty-eight to seventy-two hours after transfection, SPINK2 expression was analyzed by qPCR and Western blotting.
4
siRNA Silencing of Actin Dynamics
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Silencer Select (21 nt) siRNAs were purchased from Ambion/Lifetechnologies: siRNA1 (s34736): sense sequence 5’-CCAUGAAGGCUUUCCGGGAtt-3’); siRNA2 (s230622): sense sequence 5’-GCAUUGUCAUGAACGAGCUtt-3’. As a negative control, a random, non-targeting siRNA sequence was used. HeLa cells stably expressing Lifeact-GFP were seeded on coverslips or in 8-well Ibidi slides and transfected with siRNAs (30 nM) on the next day, using Oligofectamine (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Cells were incubated for 72 hr and either imaged live for CaAR after ionomycin addition (Figure 3E , Figure 3—figure supplement 1C ) or laser ablation (Figure 3—figure supplement 1D , 6C ) or fixed at various time-points after ionomycin addition and immunostained for MRTF-A (Figure 5C ).
5
Silencing β-catenin and βPix in Cancer Cells
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We transfected HT-29 and SNU-C4 cells with 50 nM siRNA targeting human β-catenin66 (link). We transfected HCT116 cells with siRNA targeting human βPix: siRNA #1, 5’-GAGCUCGAGAGACACAUGGTT-3’; siRNA #2, 5’-GGAUAUUAGUGUCGUGCAATT-3’ (Ambion)38 (link). Silencer Negative Control siRNA #1 (Invitrogen) was used as control. For shRNA experiments using HT-29 cells, we targeted human GIPZ viral particles against the following sequence: AGGATGAAGTTCAAGAATT (Thermo Scientific)38 (link). For shRNA, we used non-silencing-GIPZ lentiviral shRNAmir (Thermo Scientific) as control.
6
LNCaP Cell Transfection for PSA Assay
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LNCaP cells in steroid hormone-depleted medium without phenol-red, containing 10% CS-FBS were prepared for transfection. One × 105 LNCaP cells were re-suspended in 100 μl resuspension buffer R (Neon® Transfection System; Invitrogen, Carlsbad, CA, USA) with 2 μM siRNA for HSD3B1 (s6926; Silencer® select, Ambion) or control non-silencing siRNA (#1 siRNA; Ambion) and transfected in 100 μl Neon tip with Neon transfection system (Invitrogen) using two pulses (1250 V input pulse voltage/20 ms input pulse width). Five × 104 transfected cells in 500 μl phenol-red free medium containing 10% CS-FBS with 10 nM 3β-diol were plated each well of 24 well plate in triplicate and cultured for 3 days before Tandem-R PSA tests.
7
DLG2 and LIN7A Overexpression Plasmids
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DLG2 (NM_001364) and DLG2 (NM_001351274.2) overexpression plasmids on a backbone of pcDNA3.1/C-(K)-DYK (OHu25658D and OHuq102626D respectively) vector were purchased from GenScript. LIN7A (NM_004664) over expression plasmid on a backbone of pCMV6-AC-GFP (PS100010) was purchased from Origene (RG221902). siRNA targeting DLG2 (s4122), LIN7A (s16836) or Silencer™ Select Negative control No. 1 siRNA (4390843) were purchased from Ambion (Thermo Fischer Scientific). SKNAS and HEK293 cells were grown to 80% confluence and subsequently transfected with; DYK-tagged DLG2 isoform 7, DYK-tagged DLG2 isoform 2, combined with GFP-tagged LIN7A, empty vector “mock” (pCMV6-Ac-GFP), si-LIN7A or scrambled negative control “mock”. 100 ng of DNA or 10 pmol siRNA was complexed with 0.3 µl of Lipofectamine 2000 according to the Lipofectamine 2000 reagent forward transfection protocol (Invitrogen; Thermo Fisher Scientific, Inc.).
8
CDCA2 Knockdown and Overexpression Assays
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Negative control (NC) and small interfering RNAs (siRNAs) of CDCA2 were constructed by GenePharma Corporation (Shanghai, China). The siRNA sequences of CDCA2 were as follows: siRNA1, 5′-CACCUGCCUUUCUAAAUAUTT-3′; siRNA2, 5′-GGGCAAAGGAUCAAGUGAUTT-3′; siRNA3, 5′-CUGCCUUGGAAAGGAUUGATT-3′. Transfection was performed with Lipofectamine 3000 (Invitrogen) following the manufacturer’s instructions. The assays were performed 48 h after transfection to assess the knockdown efficiency. A CDCA2 inhibitor lentivirus (shCDCA2) was then constructed according to siRNA1. To upregulate the expression of CDCA2 in DLD-1 cells, mammalian expression plasmids (pReceiver-M02-CDCA2) designed to specifically express CDCA2 were obtained from GeneCopoeia (Rockville, MD, USA).
9
Silencing Human Igγ Constant Region
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Synthetic siRNA targeting the human Igγ chain constant region and control siRNAs were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). The siRNA sequences were as follows, siRNA-1, 5′-GCA AGG AGU ACA AGU GCA ATT-3′, siRNA-2, 5′-CCG GAG AAC AAC UAC AAG ATT-3′, siRNA-3, 5′-CAC AAC CAC UAC ACA CAG ATT-3′, siRNA-positive control, 5′-UGA CCU CAA CUA CAU GGU U-3′, siRNA-negative control, 5′-UUC UCC GAA CGU GUC ACG UTT-3′. Lipofectamine 3000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to transfect the siRNAs into the podocyte cell line. Following transfection, which was conducted according to the manufacturer's protocol, the knockdown efficiency was detected by western blotting. Approximately 3×105 cells/well were cultured in complete medium with a final siRNA concentration of 50 nM. The cells were transfected at 37°C for 48 h and subsequently collected for further experiments.
10
siRNA Knockdown of Hepcidin in Cardiomyocytes
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Two pairs of small-interfering RNAs (siRNA1 and siRNA2) were synthesized by Invitrogen Life Technology (Invitrogen). siRNA2 was employed to knock-down hepcidin levels and siRNA1 was injected s the negative control. Cultured human cardiomyocytes were transfected with each siRNA (20 μM) using Lipofectamine RNAiMAX (Invitrogen) as per the manufacturer’s instructions. After 48 h, protein was extracted for western blot analysis.
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