GPR109a-specific siRNA1 (CGATGTTAATCAAGAAGCA), siRNA2 (GTAGCTTCAGCAT CTGCAA), and negative control siRNA (siCtrl) (RiboBio, Guangzhou, china) were used to knockdown GPR109a. The si-GPR109a and siCtrl were transfected into IPEC-J2 using lipofectamine 3000 (Invitrogen, USA) following the manufacturer’s procedures.
Sictrl
Manufactured by RiboBio
Sourced in China
SiCtrl is a laboratory equipment used for controlling and monitoring silica-based materials. It provides precise control over the synthesis and characterization of silica-based materials in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (9 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (4 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions)
Sictrl, by Thermo Fisher Scientific (2 mentions)
Si nc, by RiboBio (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Goscript reverse transcription system, by Promega (1 mentions)
Effective sirna oligonucleotides, by RiboBio (1 mentions)
Sitak1, by RiboBio (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Itaq universal sybr green supermix, by Bio-Rad (1 mentions)
Interferin, by Polyplus Transfection (1 mentions)
Si nc, by Thermo Fisher Scientific (1 mentions)
Sirna oligonucleotides, by RiboBio (1 mentions)
Mir 944 mimic, by GenePharma (1 mentions)
Si igf1r, by RiboBio (1 mentions)
Mir nc, by GenePharma (1 mentions)
Inhibitor nc, by GenePharma (1 mentions)
Hieff trans liposomal transfection reagent kit, by Yeasen (1 mentions)
Puromycin, by Solarbio (1 mentions)
22 protocols using sictrl
1
Silencing GPR109a in IPEC-J2 Cells
2
Targeting TAK1 and NRF2 with siRNA
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Effective siRNA oligonucleotides targeting TAK1 and NRF2 were obtained from RiboBio with the following sequences:
siTAK1-#1: GGAGTGGCTTATCTTCACA;
siTAK1-#2: GGCTTATCTTACACTGGAT;
siNRF2-1#: GAGAAAGAATTGCCTGTAA;
siNRF2-2#: GCTACGTGATGAAGATGGA;
siNRF2-3#: GCCCTCACCTGCTACTTTA.
The negative control (siCtrl) was nonhomologous to any human genome sequence and purchased from RiboBio Co., Ltd. Predetermined cells were grown in 6-well plates for 12 h and then transfected with 20 nM siRNA mixed with 5 μL of Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions. Thirty-six to 48 h after transfection, the cells were harvested for further analysis.
Total RNA was extracted using TRIzol (Life Technology), reverse transcribed into cDNA using the GoScriptTM Reverse Transcription System (Promega) and analyzed by real-time qPCR on a BIO-RAD Real-time PCR machine using iTaq™ Universal SYBR® Green Super mix (Bio–Rad). The results were normalized to β-actin, and relative values were calculated using the 2[-(CT gene -CT reference)] method. The gene-specific primers used for qPCR are listed in Supplementary Table3 .
siTAK1-#1: GGAGTGGCTTATCTTCACA;
siTAK1-#2: GGCTTATCTTACACTGGAT;
siNRF2-1#: GAGAAAGAATTGCCTGTAA;
siNRF2-2#: GCTACGTGATGAAGATGGA;
siNRF2-3#: GCCCTCACCTGCTACTTTA.
The negative control (siCtrl) was nonhomologous to any human genome sequence and purchased from RiboBio Co., Ltd. Predetermined cells were grown in 6-well plates for 12 h and then transfected with 20 nM siRNA mixed with 5 μL of Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions. Thirty-six to 48 h after transfection, the cells were harvested for further analysis.
Total RNA was extracted using TRIzol (Life Technology), reverse transcribed into cDNA using the GoScriptTM Reverse Transcription System (Promega) and analyzed by real-time qPCR on a BIO-RAD Real-time PCR machine using iTaq™ Universal SYBR® Green Super mix (Bio–Rad). The results were normalized to β-actin, and relative values were calculated using the 2[-(CT gene -CT reference)] method. The gene-specific primers used for qPCR are listed in Supplementary Table
3
Knockdown of Foxf1 Gene Expression
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Foxf1-specific siRNAs (GTGTGACCGAAAGGAGTTT for human and GATCCGGCTAGCGAGTTTA for mouse) and negative control siRNA (siCtrl) (RiboBio, Guangzhou, China) were used to knockdown Foxf1. Transfection of siRNA oligonucleotides was performed using Lipofectamine RNAimax (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Foxf1 expression was determined by quantitative reverse transcription PCR (qRT-PCR), western blotting (WB), and immunofluorescence (IF). Transfected cells were passaged and used for downstream analyses.
4
Snail Gene Silencing Protocol
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siRNA duplex oligonucleotides targeting human Snail mRNA (sense: 5′-GGACACAGGUUCUGAAACA-3′ and anti-sense: 5′-CCUGUGUCCAAGACUUUGU-3′) and the sequence of si-negative control (si-ctrl) were designed by RiboBio (Guangzhou, China). pcDNA.3-Snail was a kind gift from Dr Ming Li (Sun Yat-Sen University, Guangzhou, China). Oligonucleotide transfection was performed with Lipofectamine 2000 reagent (Thermo Fisher Scientific).
5
siRNA-mediated Knockdown of LncRNAs
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Three independent siRNAs against TUC339 and lincRNA-VLDLR along with unrelated control siRNA (si-ctrl) were purchased from Ribobio (Guangzhou, China). THP-1 cells were transfected with 50 nM siRNA to lncRNA or si-ctrl using INTERFERin (Polyplus-transfection SA, Illkirch-Graffenstaden, France) for 48 h before further experiments. si-TUC339-1 was used in functional studies.
6
Overexpression and Silencing of SENCR in VSMCs
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Full length mouse SENCR cDNA was cloned into pcDNA-3.1 vector and stored in -20°C. Small interfering RNA (siRNA) against SENCR (si-SENCR) or siRNA negative control (si-NC, si-Ctrl) were designed and purchased from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). Next, pcDNA-SENCR (2 μg), empty vector (2 μg), si-SENCR (50 nM), and si-NC (50 nM) were transfected into Ang-II-induced VSMCs using Lipofectamine® 2000 (Invitrogen).
7
Targeted siRNA Knockdown Assay
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Three different siRNAs, namely anti-RACGAP1 (si-RACGAP1-1/-2/-3) and control siRNA (si-Ctrl), were purchased from Ribobio (Guangzhou, Guangdong, China). Cells were transfected with siRNAs or si-Ctrl at a concentration of 100 nM using Lipofectamine RNAiMax (Thermo Fisher Scientific) for 6–8 h according to manufacturer’s instructions. The cells were then cultured with complete medium at 37℃ in a humidified atmosphere with 5% CO2 for the indicated time points prior to use for experiments.
8
Plasmid and siRNA Transfection in Cell Culture
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The cells were cultured in a 6-well or 24-well plate for 24 hours and then were transfected with plasmids or siRNAs. All transfections were performed using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. siRNA oligonucleotides, including targeting HULC (or CLOCK) and a nonspecific scrambled control (si-Ctrl), were synthesized by RiboBio (Guangzhou, China). The siRNA duplexes sequences are all listed in Supplementary Table S1 .
9
IGF-1R Regulation via miR-944 and Plasmid Transfection
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miR-944 mimics, a miR-944 inhibitor, negative control miRNA mimics (miR-NC), and a negative control miRNA inhibitor (NC inhibitor) were provided by Shanghai GenePharma Co., Ltd. (Shanghai, China). A small interfering RNA (siRNA) used to knockdown IGF-1R expression (si-IGF-1R) and a control siRNA (si-ctrl) were chemically synthesized by Guangzhou RiboBio Co., Ltd. (Guangzhou, China).
The pcDNA3.1-IGF-1R (pc-IGF-1R) plasmid was employed to increase the expression of IGF-1R and an empty pcDNA3.1 plasmid was used as a control. Plasmids were chemically synthesized by the Chinese Academy of Sciences (Changchun, China).
For transfection, cells were plated into 6-well plates. Transfection was performed when the cells had grown to approximately 60–70% density. Lipofectamine™ 2000 (Invitrogen; Thermo Fisher Scientific) was used for transfections and assays were performed according to the product specifications. After 8 h of culture, culture medium was replaced with fresh DMEM containing 10% FBS.
The pcDNA3.1-IGF-1R (pc-IGF-1R) plasmid was employed to increase the expression of IGF-1R and an empty pcDNA3.1 plasmid was used as a control. Plasmids were chemically synthesized by the Chinese Academy of Sciences (Changchun, China).
For transfection, cells were plated into 6-well plates. Transfection was performed when the cells had grown to approximately 60–70% density. Lipofectamine™ 2000 (Invitrogen; Thermo Fisher Scientific) was used for transfections and assays were performed according to the product specifications. After 8 h of culture, culture medium was replaced with fresh DMEM containing 10% FBS.
10
Transient and Lentiviral Transfection of SRSF9, DSN1, and METTL3
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For transient transfection, small interfering RNAs (siRNAs) directed against SRSF9 (#SIGS0008682-4, RIBOBIO, Guangzhou, China), DSN1 (#SIGS0013609-1, RIBOBIO), METTL3 (#SIGS00056532-1, RIBOBIO) and negative control RNAs (si-ctrl) were synthesized by RIBOBIO Company (Guangzhou, China). Transient transfection was performed using a Hieff Trans™ Liposomal Transfection Reagent kit (#40802, Yeasen, Shanghai, China) in accordance with the standard protocol. Cells were collected after 24 h for qRT-PCR and after 48 h for Western blotting and functional studies.
For lentiviral transfection, Flag-SRSF9 (ov-SRSF9), empty vector (Vector), shSRSF9 (sh1, sh2), and shNC were purchased from GeneChem Company (Shanghai, China). Caco2 cells and HT29 cells were used to establish stable SRSF9 overexpression models, and HCT116 cells and LOVO cells were used in the stable SRSF9 knockdown experiments. According to the manufacturer’s instructions, 4 × 104 cells per well were seeded and transfected with the indicated lentiviruses. The infected cells were screened using 5 μg/mL puromycin (Solarbio, Beijing, China) for 1 week or longer, and transfection efficiency was determined by qRT-PCR and Western blotting analysis.
For lentiviral transfection, Flag-SRSF9 (ov-SRSF9), empty vector (Vector), shSRSF9 (sh1, sh2), and shNC were purchased from GeneChem Company (Shanghai, China). Caco2 cells and HT29 cells were used to establish stable SRSF9 overexpression models, and HCT116 cells and LOVO cells were used in the stable SRSF9 knockdown experiments. According to the manufacturer’s instructions, 4 × 104 cells per well were seeded and transfected with the indicated lentiviruses. The infected cells were screened using 5 μg/mL puromycin (Solarbio, Beijing, China) for 1 week or longer, and transfection efficiency was determined by qRT-PCR and Western blotting analysis.
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