siRNA was used to knock down ITGA7 expression. ITGA7 siRNA and nonsense siRNA were designed and synthesized by Guangzhou RiboBio Co., Ltd. ITGA7 siRNA (80 nM) and nonsense siRNA were transfected into Huh7 cells and SNU449 cells using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions for 6 h at 37˚C. Subsequently, cells transfected with ITAG7 siRNA were considered ITGA7-knockdown (KD) cells, and cells transfected with nonsense siRNA were marked as control cells. At 24 h after transfection, lTGA7 mRNA and protein expression levels were determined by RT-qPCR and western blotting; cell apoptosis was detected by an annexin V/propidium iodide (AV/PI) assay at 48 h, expression of apoptosis-related protein cleaved caspase 3 was detected by western blotting, and cell migration and invasion abilities were assessed by wound scratch and Transwell assays, respectively. Cell viability was assessed using a Cell Counting Kit-8 (CCK-8) assay at 0, 24, 48 and 72 h. Additionally, whether the mRNA and protein expression of E-cadherin, vimentin, N-cadherin and α-SMA were regulated by ITGA7 was determined by RT-qPCR and western blotting at 24 h after transfection. In addition, the sequences of ITGA7 siRNA were as follows: Forward, 5'-GCAUCAAGAGCUUCGGCUATT-3' and reverse, 5'-UAGCCGAAGCUCUUGAUGCTT-3'.
Nonsense sirna
Manufactured by RiboBio
Sourced in China
Nonsense siRNA is a laboratory reagent used in gene knockdown experiments. It is a synthetic small interfering RNA (siRNA) molecule that does not target any known gene in the organism under study. Nonsense siRNA serves as a control to establish baseline gene expression levels and evaluate the specificity of target gene silencing by other siRNA molecules.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Gel extraction kit, by Takara Bio (1 mentions)
In vivo sirna, by RiboBio (1 mentions)
Tgfbr2 sirna, by RiboBio (1 mentions)
Interleukin 1β il 1β, by Merck Group (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Pcdna3.1 vector, by Thermo Fisher Scientific (1 mentions)
Sirna duplexes, by RiboBio (1 mentions)
7 protocols using nonsense sirna
1
ITGA7 Knockdown Impacts Cellular Processes
2
Cloning and Silencing of PRMT5 Regulatory Factors
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The PRMT5-minigene, a segment of PRMT5 including the region from exon 1 to exon 4, was amplified by polymerase chain reaction (PCR) from genomic DNA. The human HNRNPH1 and SRSF3 open reading frames (ORFs) were amplified by RT-PCR. The primers are listed in Supplementary Table S1 . Next, the PCR products were gel-purified with the Gel Extraction Kit (Takara, Beijing, China) and then in-fusion cloned into the pcDNA 3.1-FLAG vector, which had been digested by BamHI and XhoI restriction endonucleases (NEB, Beijing, China) using the ExonArt Seamless Cloning and Assembly Kit (Exongen, Chengdu, China). The full-length PRMT5 (Myc-PRMT5L) and short-length PRMT5 ( Myc-PRMT5S, which was translated from PRMT5-ISO5) plasmids were generous gifts from Prof. Jiuyong Xie (Department of Physiology & Pathophysiology, University of Manitoba) as described [46 (link)].
The normal siRNA and In vivo siRNA (2′ O-methyl + 5′ cholesterol-modified) against HNRNPH1 or SRSF3 and nonsense siRNA (RiboBio, Guangzhou, China) were dissolved in rNase-free water for cell transfection or phosphate buffered saline (5 nmol siRNA with 50 μL PBS) for intratumoral injection. The sequences of si-HNRNPH1 and si-SRSF3 are shown inSupplementary Table S1 [47 (link),48 (link)].
The normal siRNA and In vivo siRNA (2′ O-methyl + 5′ cholesterol-modified) against HNRNPH1 or SRSF3 and nonsense siRNA (RiboBio, Guangzhou, China) were dissolved in rNase-free water for cell transfection or phosphate buffered saline (5 nmol siRNA with 50 μL PBS) for intratumoral injection. The sequences of si-HNRNPH1 and si-SRSF3 are shown in
3
p27 Knockdown in MDA-MB-231 Cells
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p27 siRNA (siG10514125225‐1‐5) and nonsense siRNA was obtained from RiboBio (Guangzhou RiboBio Co., China). MDA‐MB‐231 cells (3×105 cells per well) were plated in 6 wells and were transiently transfected with 2 µg of siRNAs plus 4 µL Lipofectamine 2000 according to the manufacturer's instruction. 72 h later, cells were harvested.
4
Silencing AQP4 Expression in PC12 Cells
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At first, the factor information of AQP4 was gathered from NCBI. Then, three siRNAs specifically inhibited AQP4 and one nonsense siRNA as a negative management area unit of measurement designed and purchased from Ribobio Company. Target ribonucleic acid series of fragment one (F1) is CCAAGTCCGTCTTCTACAT; fragment two (F2): CAGGTGCACTTTACGAGTA; fragment three (F3): CAGCATGAATCCAGCTCGA. In brief, once the cells reached 80% confluence, contemporary average holding siRNA portions were inserted into cells in accordance with the manufacturer's protocol from Ribobio Company. The PC12 cells were haphazardly divided into the normal group, NC group, chemical agent group, F1 group, F2 group, and F3 group. RT‐qPCR was performed to observe the consequences of siRNAs, and the most effective siRNA fragment was used for the later experiment.
5
Chondrocyte Transfection and Stimulation
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The coding sequence of PART‐1 and TGFBR2 was subcloned into the pcDNA3.1 (+) vector (Invitrogen). PART‐1 siRNA, TGFBR2 siRNA, nonsense siRNA, miR‐590‐3p mimic, miR‐590‐3p inhibitor and respective controls were purchased from Guangzhou Ribobio company. Chondrocytes were transfected with the above plasmids, siRNAs or miRNAs using Lipofectamine 3000 (Invitrogen), and transfected chondrocytes were collected for in vitro assays at 24‐72 hours after transfection. In some experiments, the cells were stimulated with 5 ng/mL interleukin 1β (IL‐1β; Sigma‐Aldrich) for 24 hours.
6
Inhibiting ADAM17 in CRC Exosomes
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To inhibit ADAM17 expression in CRC cell-derived exosomes, ADAM17-directed siRNAs were employed. Human ADAM17-specific siRNA (5′-TGAGGCAGTCTCTCCTATTCCTGACCAGC-3′) and nonsense siRNA (5′-TGACCACCCTGACCTACGGCGTGCAGTGC-3′) were obtained from RiboBio (Guangzhou, China). Cells were transfected with ADAM17-siRNA, and nonsense (scrambled) control siRNA using a liposome-based method for 48 h; then, the exosomes were collected for subsequent analyses.
7
Transient Transfection and Alix Knockdown
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Transient transfection of cells was performed by mixing the plasmids and Neofect™ DNA transfection reagent according to the manufacturer’s instructions. To inhibit the expression of Alix, siRNA duplexes targeting the sequence (GGCACAGGCTCAAGAAGTA) of Alix were used (RiboBIO, Guangzhou, China). Briefly, 3 × 105 cells per well of a 12-well plate were transfected with 15 pmol siRNA and 4.5 μl Lipofectamine RNAiMAX transfection reagent (13778, Thermo Fisher Scientific, Waltham, MA, USA) according to the protocol of the supplier. As a control, a nonsense siRNA with no known homology to the mammalian gene was used (RiboBIO, Guangzhou, China). After 36 h, cells were retransfected with plasmid DNA with Neofect™ DNA transfection reagent and harvested after an additional 48 h.
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