For AAV transduction in primary neurons, the viral particles were added to neurobasal medium at a multiplicity of infection of 1 × 106 on the second day of plating. The neurons were analyzed on 14 days in vitro (DIV). The transduction efficiency was observed at > 90%. For miRNA transduction in primary neurons, the neurons were transfected on DIV 12. According to the manufacturer’s instructions, Lipo3000 (L3000008, Invitrogen) was used to transfect 100 nm of miRNA agomir or antagomir into each well of cells using Opti-MEMTM I Reduced Serum Medium (31985062, Thermo Fisher). The cells were examined 48 h after miRNA agomir or antagomir transfection. The miRNA agomir, antagomir, negative control NC agomir, and NC antagomir were purchased from RiboBio (Guangzhou, China).
Mirna agomir
Manufactured by RiboBio
Sourced in China
The MiRNA agomir is a synthetic, double-stranded RNA molecule designed to mimic the function of a specific microRNA (miRNA) in biological systems. It serves as a tool for researchers to study the role and function of miRNAs in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Antagomir, by RiboBio (1 mentions)
Antagomir nc, by RiboBio (1 mentions)
Opti memtm 1 reduced serum medium, by Thermo Fisher Scientific (1 mentions)
Lipo3000, by Thermo Fisher Scientific (1 mentions)
Pgl6 ta vector, by Beyotime (1 mentions)
Mpk5000, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Pmirglo vector, by Promega (1 mentions)
Mir 145 5p agomir, by RiboBio (1 mentions)
3 protocols using mirna agomir
1
Efficient AAV and miRNA Transduction in Primary Neurons
2
Characterization of Pcsk9 3'UTR Regulation
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A 384 bp fragment of the Pcsk9 3’UTR (containing 2 predicted positions) was amplified by PCR using mouse genomic DNA as a template, and cloned into the pmir-GLO vector (Promega). Overlap PCR was used to construct 3′-UTR mutant reporter plasmid. The sequences of wild-type and mutant 3′-UTR were confirmed by sequencing. HEK 293T cells were seeded in 24-well plates and transfected with a mixture of pmir-GLO-Pcsk9 3’UTR (wild-type or mutant) and miRNA agomir (RiboBio, China) following the recommended protocol for the Lipofectamine 2000 transfection system (Invitrogen). Luciferase activity was assayed 2 days after transfection using the POLARstar Omega. TCF/LEF response element was cloned into the pGL6-TA vector (Beyotime Biotechnology). For transfecting 14837CAFs with TCF/LEF luciferase reporter plasmid and pRL-TK vector, neon transfection system (Invitrogen, MPK5000) was used following the manufacturer’s instructions. Then, 14837CAFs were cultured with 14837T-CM or 15376T-CM in the presence or absence of 200 ng/ml r-mWnt5a. After 3 days, luciferase activity was assayed. Firefly luciferase activity was normalized to Renilla luciferase activity for each sample.
3
TNBS-Induced Colitis Model in Mice
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The TNBS-induced colitis model was conducted in male BALB/c mice (week age: 6-8 w; weight: 20-22 g, Nanjing Biomedical Research Institute of Nanjing University, Nanjing, China), and raised under specific pathogen-free conditions in the Animal Experimental Center of our hospital. This colitis mouse model was established following a previously published protocol.19 (link),20 (link) Experimental groups were pre-sensitised with 1% (w/v) TNBS acid (Sigma, St. Louis, MO, USA) for 7 days, and then challenged with 100 μL 2.5% TNBS mixture (one volume 5% TNBS in one volume of absolute ethanol) on day 8 by enema, whereas the control group were administered with same volume of 50% ethanol solution. After another 7 days, all mice were sacrificed for further research. Furthermore, the TNBS-induced colitis model was also established in Cldn8−/− mice to demonstrate the role of the miR-145/SOX9/CLDN8 pathway in colitis initiation.
MiRNA agomir (RiboBio, Shanghai, China) was administered to TNBS-induced colitis mice to observe the effect of miR-145-5p on regulating the expression of Sox9 in vivo. miR-145-5p agomir and its corresponding negative control at a dose of 10 mg/kg were administered to TNBS-induced colitis mice via intraperitoneal injection on days 8, 9, and 10. In addition, the sequences of miR-145-5p agomir could be required from RiboBio (Shanghai, China).
MiRNA agomir (RiboBio, Shanghai, China) was administered to TNBS-induced colitis mice to observe the effect of miR-145-5p on regulating the expression of Sox9 in vivo. miR-145-5p agomir and its corresponding negative control at a dose of 10 mg/kg were administered to TNBS-induced colitis mice via intraperitoneal injection on days 8, 9, and 10. In addition, the sequences of miR-145-5p agomir could be required from RiboBio (Shanghai, China).
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