SNFB cells were seeded into a 24-well plate at a concentration of 1 × 105/well, and incubated for 24 hours. Then, 1.25 μL of the siRNA of Act B (20 μM) (siRNA-Activin B kit, Guangzhou RiboBio Co., Ltd., Guangzhou, China) was diluted with 50 μL 1× riboFECTTMCP buffer (riboFECTTMCP transfection reagent, Guangzhou RiboBio Co., Ltd., Guangzhou, China) and incubated at room temperature for 5 minutes. Then, 5 μL riboFECTTMCP reagent (riboFECTTMCP transfection reagent, Guangzhou RiboBio Co., Ltd., Guangzhou, China) was added and mixed. The mixture was mixed with 443.75 μL cell culture media, added to the cells and incubated for 48 hours. The transfection efficiency was observed under an inverted fluorescence microscope (Olympus Co., Tokyo, Japan).
Ribofecttm cp transfection reagent
Manufactured by RiboBio
Sourced in China
RiboFECTTM CP Transfection Reagent is a cationic polymer-based transfection reagent designed for efficient delivery of nucleic acids, such as plasmid DNA and siRNA, into a variety of mammalian cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Pmir report luciferase vector, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter gene assay system, by Promega (1 mentions)
Inverted fluorescence microscope, by Olympus (1 mentions)
Sirna oligonucleotides, by RiboBio (1 mentions)
H9c2 cell, by American Type Culture Collection (1 mentions)
Mimic nc, by RiboBio (1 mentions)
Double luciferase reporter gene detection kit, by Beyotime (1 mentions)
Agomir, by RiboBio (1 mentions)
Agomir control, by RiboBio (1 mentions)
Mirna mimic, by RiboBio (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Beta actin, by Bioss Antibodies (1 mentions)
Ripa lysate, by Solarbio (1 mentions)
Bca protein concentration determination kit, by Solarbio (1 mentions)
Firefly luciferase reporter gene assay kit, by Beyotime (1 mentions)
Mir 126 3p antagomir, by RiboBio (1 mentions)
13 protocols using ribofecttm cp transfection reagent
1
Silencing Activin B in SNFB Cells
2
Characterizing Snail 3'UTR-miR-30b Interaction
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The 3'UTR sequence of human Snail gene containing predicted binding site for miR-30b was amplified by PCR using PANC-1 cell genomic DNA as a template. PCR primer sequences are 5'-CCC AAG CTT GGG GGG ACT GTG AGT AAT GGC TG-3' (forward) and5'-CGA GCT CGA ACG TTT CCT TGA TAC AAA ATG T-3' (reverse). Snail wild-type (WT-3'UTR) vector was constructed by inserting the PCR fragment into pMIR-REPORT luciferase vector (Ambion, USA). Mutant vector (MUT-3'UTR) was constructed using an overlap extension method. Cells were planted into 24-well plate at 50-60% confluency. 24 h later, cells were transfected with pRL-TK vector (20ng) and WT-3'UTR or MUT-3'UTR vectors (180ng), along with miR-30b mimic or NC mimic at a concentration of 100 nM using riboFECTTM CP Transfection Reagent (Ribo-Bio Co Ltd., China). Each group is composed of three duplicate wells. 24 hours after transfection, the relative firefly luciferase activity (normalized to Renilla luciferase activity) was measured using a dual-luciferase reporter gene assay system (Promega, USA), and results were depicted as fold changes relative to respective controls.
3
Modulating circCacna1c in ISO-induced H9c2 cells
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The H9c2 cells (ATCC, Manassas, VA, USA) were cultured in high-glucose Dulbecco’s modified Eagle’s medium (HyClone, Cat# SH30022.01, Logan, UT, USA) containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in an incubator with 5% CO2. For the ISO-induced cell model, the H9c2 cells were incubated with 30 μM ISO in a serum-free medium for 36 h.
Small interfering RNA (siRNA) oligonucleotides specific to circCacna1c and negative control siRNA (si-NC) were designed and synthesized by RiboBio (Guangzhou, China). The siRNA sequence for circCacna1c was 5′-UCCCAUAGUUGGAACCAGG-3′. The mmu-miR-29b-2-5p mimic (miR-mimic) and their corresponding negative controls (NC-mimic) were purchased from RiboBio. The sequence for miR-mimic was 5′-CUGGUUUCACAUGGUGGCUUAGAUU-3′. The siRNAs or miRNA mimics were transfected into H9c2 cells using the riboFECTTM CP transfection reagent (RiboBio, Cat# C10511-1, Guangzhou, China), according to the manufacturer’s instructions.
Small interfering RNA (siRNA) oligonucleotides specific to circCacna1c and negative control siRNA (si-NC) were designed and synthesized by RiboBio (Guangzhou, China). The siRNA sequence for circCacna1c was 5′-UCCCAUAGUUGGAACCAGG-3′. The mmu-miR-29b-2-5p mimic (miR-mimic) and their corresponding negative controls (NC-mimic) were purchased from RiboBio. The sequence for miR-mimic was 5′-CUGGUUUCACAUGGUGGCUUAGAUU-3′. The siRNAs or miRNA mimics were transfected into H9c2 cells using the riboFECTTM CP transfection reagent (RiboBio, Cat# C10511-1, Guangzhou, China), according to the manufacturer’s instructions.
4
Evaluating miR-335-5p Binding to JAG1
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Wild‐type (wt) and mutant type (mut)‐3’‐UTR of JAG1 were amplified. Double enzyme digestion was conducted using XhoI and NotI, and the psi‐Cpsi‐CHECK‐2 vector (Promega Corporation, Madison, WI, USA) was connected with T4DNA ligase to construct JAG1‐wt and JAG1‐mut plasmids. According to the instructions of RiboFECTTMCP transfection reagent (Guangzhou RiboBio Co., Ltd., Guangzhou, Guangdong, China), 100 ng JAG1‐wt or JAG1‐mut was co‐transfected with miR‐335‐5p NC or miR‐335‐5p mimic in a respective manner into 293 T cells and cultured in the 24‐well plate for 48 h. Three replicates were prepared. The experiment was repeated for 3 times. In accordance with the instructions of double luciferase reporter gene detection kit (Beyotime Institute of Biotechnology, Shanghai, China), cells were lysed for 15 minutes. Next, the luminous value was tested by Tecan Infinire@200 Pro, a multifunctional enzyme marker instrument, and standardization was performed with stably expressed renilla luciferase value to test luciferase activity.
5
Modulation of GAS5 and Sox9 Expression
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The siRNAs (GAS5 (Norway rat) si-RNA, Sox9 (Norway rat) si-RNA), overexpression plasmids (pZdonor-CMV-Gas5 (rat, NR-002704)-SV40 promoter-neo, pZdonor-CMV-Sox9 (rat, NM-080403)-SV40 promoter-neo), and corresponding negative controls (si-nc Negative control, oe-nc pZdonor-CMV-MCS-SV40 promoter-neo) of GAS5 and Sox9 were obtained from SyngenTech (Beijing, China). The cells were seeded in 6- or 24-well plates for 12 h before the experiment. Transfection was performed using the Polyplus jetPRIME transfection kit (Polyplus Transfection, France) according to the manufacturer’s instructions.
MiR-1912-3p mimics and inhibitors and the corresponding negative controls were purchased from Guangzhou RiboBio Co., Ltd. Transfection was performed using the RiboFECTTMCP transfection reagent (RiboBio Co. Ltd., Guangzhou, China) according to the manufacturer’s protocol.
MiR-1912-3p mimics and inhibitors and the corresponding negative controls were purchased from Guangzhou RiboBio Co., Ltd. Transfection was performed using the RiboFECTTMCP transfection reagent (RiboBio Co. Ltd., Guangzhou, China) according to the manufacturer’s protocol.
6
siRNA Transfection of Lung Cell Lines
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Beas2B cells and A549 cells were obtained from Zhong Qiao Xin Zhou Biotechnology. Beas2B cells were cultured in complete bronchial epithelial cell medium (Zhong Qiao Xin Zhou Biotechnology, China), while A549 cells were cultured in RPMI 1640 with 10% FBS and 1% penstreptomycin in a 37°C incubator under 5% CO2.
Beas2B and A549 cells were separately seeded in six-well plates at a density of 3 × 105 cells/well and then transfected for 24 h with 50 nM GLCCI1 siRNA (si-h-GLCCI1_001: GCTCTATGATCGTGATAAA) and with 50 nM negative control (NC) siRNA (Ribobio Co., Ltd., China) with ribo FECTTMCP transfection reagent (Ribobio Co., Ltd., Chia) according to the manufacturer's instructions.
Beas2B and A549 cells were separately seeded in six-well plates at a density of 3 × 105 cells/well and then transfected for 24 h with 50 nM GLCCI1 siRNA (si-h-GLCCI1_001: GCTCTATGATCGTGATAAA) and with 50 nM negative control (NC) siRNA (Ribobio Co., Ltd., China) with ribo FECTTMCP transfection reagent (Ribobio Co., Ltd., Chia) according to the manufacturer's instructions.
7
Icaritin Modulation of STAT3 Signaling
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2 × 105 U266 cells were plated onto a 6-well culture plate in 2 ml complete medium, siRNA oligonucleotides targeting STAT3 gene (CCGTGGAACCATACACAAA) and negative control siRNA (Guangzhou Ribobio CO., LTD, China) were transfected at a final concentration of 50 nM by using ribo FECTTM CP transfection reagent (Guangzhou Ribobio CO., LTD, China). After 24 hours, the cells were exposed to DMSO or icaritin (16 μM) for 48 hours, cells were collected and cellular proteins were extracted for western blotting assay.
8
Targeting miR-181a-5p and Endocan in HRECs
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Control and endocan‐targeting small interfering RNAs (siRNAs) were synthesized by GenePharma Co. Ltd. (Shanghai, China). AgomiR, AgomiR control, miRNA mimic, inhibitor, and nontargeting control oligonucleotides (RiboBio, Guangzhou, China) were transfected into HRECs using riboFECTTM CP Transfection Reagent (RiboBio). Briefly, cells were seeded onto sixwell plates (Corning Inc., Corning, NY) at a density of 5 × 104 cells/ml, transfected with miR‐181a‐5p mimic, miR‐181a‐5p inhibitor, endocan siRNA, or negative control using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. After 48 hr, cells were treated with rhVEGFA (20 ng/ml) for the indicated time period. Specific sequences are listed in Table S1.
9
Transfection of miR-30b Mimic in PCSCs
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MiR-30b mimic and NC mimic were designed and synthesized by Ribo-Bio Co Ltd (Guangzhou, China). PCSCs were seeded in 6 well plates and incubated overnight, reaching 40% to 50% confluence, and then transiently transfected with 100 nM miR-30b mimic or NC mimic using riboFECTTM CP Transfection Reagent (Ribo-Bio Co Ltd., China). 24 h after transfection, cells were harvested for subsequent experiments.
10
Bovine Herpesvirus Type 1 Cell Study
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Bovine herpesvirus type 1 (BHV−1) and MDBK were stored in the cell biology laboratory of Heilongjiang Bayi Agricultural University (Daqing, China). The Ribo FECTTMCP transfection reagent and Ribo lncRNA smart silencer interfering plasmid were designed and synthesized by Guangzhou RiboBio. RNA extraction, cDNA reverse transcription, and fluorescence quantitative PCR kits were all purchased from Nanjing Vazyme Biotechnology. A BCA protein concentration determination kit and RIPA lysate were purchased from Solarbio. Beta actin and sheep anti−mouse IgG−HRP antibodies were purchased from Bioss. NF−κB, JNK and IκB antibodies were purchased from Affinity Biosciences.
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