Sirna duplexes

Manufactured by RiboBio

Sourced in China

SiRNA duplexes are short, double-stranded RNA molecules designed to target and silence specific genes within cells. They function by interfering with the expression of target genes, leading to the degradation of the corresponding mRNA and the subsequent reduction in the production of the encoded proteins.

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SiRNAs (265 citations) SiRNA) duplex (6 citations) HDAC3 siRNA duplex (4 citations) SiRNA oligonucleotide duplexes (3 citations) SiRNA control duplexes (3 citations) Short-interfering RNA (siRNA) duplexes (2 citations)

40 protocols using sirna duplexes

Targeted Gene Silencing with siRNA

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Small interfering RNA (siRNA) duplexes targeting human SATB1, c-Jun, NFkB1, Sp1 sequence and scrambled siRNA were produced by Ribobio Co. Ltd. (Guangzhou, China) and their knock-down efficiency was validated by Real-time PCR and Western Blot. siRNA sequences were as follows:
SATB1, sense, 5′-GGAUAGUCUUUCUGAGCUA-3′, antisense, 5′-UAGCUCAGAAAG ACUAUCC-3′;
Sp1 si-1, sense, 5′-CCAACAGAUUAUCACAAAU-3′, antisense, 5′-AUUUGUGAUAAUCUGUUGG-3′;
Sp1 si-2, sense, 5′-GGCUGGUGGUGA UGGAAUA-3′, antisense, 5′-UAUUCCAUCACCACCAGCC-3′;
NFKB1, sense, 5′-GGGGCUAUAAUCCUGGACU-3′, antisense, 5′-AGUCCAGGAUUGUAGCCCC-3′;
c-Jun, sense, 5′-CUGCAAAGAUGGAAACGAC-3′, antisense, 5′-GUCGUUUCCAUCUUUGCAG-3′,
NC, sense, 5′-UUCUUCGAACGUGUCAC G-3′, antisense, 5′-ACGUGACACGUUCGGAGAATT-3′.

Gong J., Tu W., Han J., He J., Liu J., Han P., Wang Y., Li M., Liu M., Liao J, & Tian D. (2016). Hepatic SATB1 induces paracrine activation of hepatic stellate cells and is upregulated by HBx. Scientific Reports, 6, 37717.

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Rab7 Protein Localization Study

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Human wild-type Rab7 coding sequence was cloned and inserted into EcoRI/BamHI site of pEGFP-C1 vector downstream of the GFP sequence in the right reading frame. siRNA duplexes were purchased from RiboBio Co., Ltd. (Guangzhou, China). HeLa cells were transfected with plasmids or siRNA duplexes (10 nM) using Lipofectamine™ 2000 (Invitrogen) in accordance with the manufacturer’s instructions. Rab7 and scrambled control siRNA duplexes were as described previously by Vanlandingham and Ceresa (16 (link)) with the modifications of two deoxythymidine added to the 3′ end of each sequence to make it more stable.

HU X., YU J., ZHOU X., LI Z., XIA Y., LUO Z, & WU Y. (2014). Synergism between upregulation of Rab7 and inhibition of autophagic degradation caused by mycoplasma facilitates intracellular mycoplasma infection. Molecular Medicine Reports, 9(3), 793-800.

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Silencing HMGB1 in RAW264.7 cells

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siRNA duplexes targeting HMGB1 and negtive control (NC) were designed and synthesized by RiboBio (Guangzhou, China). Transfection was performed using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions. Briefly, 24 h before transfection, Raw264.7 cells were seeded in 6-well plates at 40% confluency, and transfected with 100 nM HMGB1 siRNA or NC siRNA for 72 h.

Wang G., Jin S., Huang W., Li Y., Wang J., Ling X., Huang Y., Hu Y., Li C., Meng Y, & Li X. (2021). LPS-induced macrophage HMGB1-loaded extracellular vesicles trigger hepatocyte pyroptosis by activating the NLRP3 inflammasome. Cell Death Discovery, 7, 337.

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Generating MMP7 and MMP9 Constructs

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The MMP7 and MMP9 constructs were generated by sub-cloning polymerase chain reaction (PCR)-amplified full-length human MMP7 or MMP9 cDNA into pcDNA3.1. For depletion of MMP7 and MMP9, siRNA sequences were cloned into GV248 to generate GV248-RNAi(s) targeting MMP7 and MMP9. siRNA duplexes were synthesized and purified by RiboBio Inc (Guangzhou, People’s Republic of China). The siRNA sequences are listed in Table S1. The PCR primers are listed in Table S2. siRNA transfection was carried out using Lipofectamine 2000 reagent (Invitrogen). Retroviral production and infection were performed according to the manufacturer’s instructions. Following selection, lung cancer cell lysates prepared from the pooled cell populations in sampling buffer were fractionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) to detect protein levels via Western blotting.

Sun Y., Chen Y., Li S., Lei Y., Xu D., Jiang N., Zhang Y., Cao J, & Ke Z. (2017). NanoVelcro-captured CTC number concomitant with enhanced serum levels of MMP7 and MMP9 enables accurate prediction of metastasis and poor prognosis in patients with lung adenocarcinoma. International Journal of Nanomedicine, 12, 6399-6412.

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siRNA Knockdown of FOXM1 and EXO1

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siRNA duplexes were prepared by RiboBio(Guangzhou,China). SiRNA The sequence of siRNAs were as follows: FOXM1 siRNA-1: 5'- GCCAAUCGUUCUCUGACAGAATT-3', siRNA-2: 5'- GGACCACUUUCCCUACUUUUUTT-3'[19] (link). EXO1 siRNA-1: 5'-CAAGCCUAUUCUCGUAUUUTT-3', siRNA-2: 5'-UAGUGUUUCAGGAUCAACAUCAUCU-3'[18] (link). The sequence of negative control (NC) was: 5′-UUCUCCGAACGUGUCACGUTT-3'. Transfection or co-transfection of siRNA and plasmid was performed according to the manufacturer's protocol of lipofectamin (Invitrogen).

Zhou J., Wang Y., Wang Y., Yin X., He Y., Chen L., Wang W., Liu T, & Di W. (2014). FOXM1 Modulates Cisplatin Sensitivity by Regulating EXO1 in Ovarian Cancer. PLoS ONE, 9(5), e96989.

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Silencing AMPK via RNAi Knockdown

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siRNA duplexes against AMPK were synthesized by RiboBio Co., Ltd (Guangzhou, China) (si-AMPK #1: sense sequence 5′-GCAGAAGTATGTAGAGCAA-3′; si-AMPK #2: sense sequence 5′-ACACATGAATGCAAAGATA-3′). The silencing of genes using 10 nM RNAi duplexes was performed with Lipofectamine^TM RNAiMAX Reagent (13778-150, Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s recommendations.

Shen J.J., Zhan Y.C., Li H.Y, & Wang Z. (2019). Ouabain impairs cancer metabolism and activates AMPK-Src signaling pathway in human cancer cell lines. Acta Pharmacologica Sinica, 41(1), 110-118.

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Establishment and Cultivation of NCTC1469 Liver Cells

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NCTC1469, a liver cell line established in the 1957 from a neonatal mouse^{36 (link)}, was obtained from the FuDan IBS Cell Center (FDCC, Shanghai, China). NCTC1469 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Corning, USA) supplemented with 10% (v/v) Horse serum (Hyclone, Logan, UT, USA), 100 units/ml penicillin and 100 μg/ml streptomycin (Gibco, USA), at 37 °C in a 5% CO₂ incubator. Culture medium was replaced every 2–3 days and cells were passaged when they reached 80% confluence. Cells were mycoplasma negative through treatment with LookOut® Mycoplasma Elimination Kit (Sigma-Aldrich). Gene silencing was achieved by transfecting predesigned siRNA duplexes (Supplementary Table 3) designed and synthesized by RiboBio (Guangzhou, China).

Liang C.Q., Zhou D.C., Peng W.T., Chen W.Y., Wu H.Y., Zhou Y.M., Gu W.L., Park K.S., Zhao H., Pi L.Q., Zheng L., Feng S.S., Cai D.Q, & Qi X.F. (2022). FoxO3 restricts liver regeneration by suppressing the proliferation of hepatocytes. NPJ Regenerative Medicine, 7, 33.

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In Vitro Evaluation of NGF siRNA Efficacy

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We evaluated the in vitro transfection efficiency of NGF siRNA sequences by measuring NGF mRNA level in Panc-1 cells. Five siRNA duplexes against human NGF and a nsRNA as controls (Supplementary Table 1) were synthesized by Ribobio (Guangzhou, China). For transfection, Panc-1 cells were plated at a density of 3 × 10⁵ cells per well in a six-well plate. When the cells were 50% confluent, siRNA duplex (100 nM for each) was mixed with 5 μl Lipofectamine 2000 Reagent (Invitrogen) and incubated with Panc-1 cells for 48 h according to the manufacturer's protocol. Each treatment was conducted at least in triplicate. The expression levels of NGF mRNA in Panc-1 cells were analysed by quantitative real-time PCR (RT–PCR). The most effective siRNA sequence that result in the greatest knockdown of NGF mRNA was selected for further study (NGF siRNA-#2).

Lei Y., Tang L., Xie Y., Xianyu Y., Zhang L., Wang P., Hamada Y., Jiang K., Zheng W, & Jiang X. (2017). Gold nanoclusters-assisted delivery of NGF siRNA for effective treatment of pancreatic cancer. Nature Communications, 8, 15130.

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Lentiviral-based overexpression and knockdown of PHF2 and CDKN2B-AS1 in EBV+ cell lines

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The lentiviruses for PHF2 and CDKN2B-AS1 overexpression and the corresponding control viruses were purchased from VectorBuilder (China). C666-1 and HK1 EBV+ cell lines were infected with recombinant lentivirus plus 5 mg/mL polybrene (VectorBuilder, China) according to the manufacturer’s instructions and were selected using 5 μg/mL puromycin (MP Biomedicals, LLC, USA).
For PHF2 knockdown, siRNA duplexes targeting PHF2 and the negative control siRNA (si-NC) were designed and synthesized by RiboBio (Guangzhou, China). For CDKN2B-AS1 knockdown, the lncRNA smart silencer (a mixture of three siRNAs and three ASOs targeting CDKN2B-AS1) and smart silencer of the negative control (ss-NC) was used (RiboBio, Guangzhou, China). Lipofectamine 3000 (Invitrogen, USA) was used for cell transfection according to the manufacturer’s instructions. The sequences of siRNA and lncRNA smart silencer are shown in Table S5. To validate the effects of overexpression and knockdown of target genes in C666-1 and HK1 EBV+ cell lines, the changes of protein abundance of PHF2 were verified by western blot. For the non-coding RNA CDKN2B-AS1, the expression changes were measured by qPCR.

Wang T.M., Xiao R.W., He Y.Q., Zhang W.L., Diao H., Tang M., Mai Z.M., Xue W.Q., Yang D.W., Deng C.M., Liao Y., Zhou T., Li D.H., Wu Y.X., Chen X.Y., Zhang J., Li X.Z., Zhang P.F., Zheng X.H., Zhang S.D., Hu Y.Z., Cai Y., Zheng Y., Zhang Z., Zhou Y., Jin G., Bei J., Mai H.Q., Sun Y., Ma J., Hu Z., Liu J., Lung M.L., Adami H.O., Ye W., Lam T.H., Shen H, & Jia W.H. (2023). High-throughput identification of regulatory elements and functional assays to uncover susceptibility genes for nasopharyngeal carcinoma. American Journal of Human Genetics, 110(7), 1162-1176.

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siRNA-Mediated Protein Knockdown in Cells

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For small interfering RNA (siRNA) experiments, siRNA duplexes were used to reduce p53, p21 and E-cadherin protein expression, respectively (Ribobio, China). Briefly, cells were trypsinized and 1×10⁶ cells were suspended in 100μl Opti-MEM or medium without FBS, then added with 5 μL of 20 μM siRNA duplexes, and electro-transfected by using Super Electroporator NEPA21 (NEPA GENE, Japan). Cells were then transferred to 35-mm tissue culture dishes with 2 mL RPMI1640 medium. The siRNA sequences for knockdown of target genes are shown in Table 2.

Dai Y., Wang L., Tang J., Cao P., Luo Z., Sun J., Kiflu A., Sai B., Zhang M., Wang F., Li G, & Xiang J. (2016). Activation of anaphase-promoting complex by p53 induces a state of dormancy in cancer cells against chemotherapeutic stress. Oncotarget, 7(18), 25478-25492.

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