P19CL6 cells were obtained from RIKEN BioResource Center (Ibaraki, Japan, # RCB2318). Cells were mycoplasma-free and were grown in Dulbecco-Modified Minimal Essential Medium supplemented with 10% fetal bovine serum. For differentiation and transient transfection, cells were plated at a density of 5.0 × 105 cells per well on a 35-mm tissue culture dish containing 25 pmol of a pool of Silencer Select Pre-Designed Tbx1 SiRNA (Life Technology) and 7.5 μl of Lipofectamine RNAiMAX Reagent (Life Technology) diluted in 500 μl of Opti-MEM Medium. Cells were incubated at 37 °C in 5% CO2. Twenty hours later, the medium was replaced with one containing 10 μM 5-Azacytidine and 1 mM TCP or saline solution for 24 h. (ref. 17 (link)). After 24 h, the cells were collected and processed for further analysis.
Silencer select pre designed tbx1 sirna
Manufactured by Thermo Fisher Scientific
Silencer Select Pre-Designed Tbx1 SiRNA is a small interfering RNA (siRNA) product designed to target the Tbx1 gene. The Tbx1 gene is involved in various cellular processes. This siRNA is intended for use in RNA interference (RNAi) studies to knockdown Tbx1 gene expression in cell lines and other research applications.
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2 protocols using silencer select pre designed tbx1 sirna
1
Tbx1 Knockdown and Cardiomyocyte Differentiation
2
Mouse Myoblast and Fibroblast Culture and siRNA Knockdown
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Mouse C2C12 undifferentiated myoblast cells obtained from the ATCC (catalog CRL-1772) were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% FBS and L-glutamine (300 μg/ml), and were free of mycoplasma. Primary mouse embryonic fibroblasts (MEFs) were isolated from Tbx1 +/-, Tbx1 -/-, and wildtype embryos at E13.5. To this end, the internal organs, head, tail and limbs were removed. Cells were cultured in Dulbecco's modified Eagle's medium with 20% FBS and 1% NEAA, and used for a maximum of 3 passages. Cells were incubated at 37°C in 5% CO 2 . For siRNA transfection, cells were seeded at 1.2×10 5 per well in six-well plates and transfected with a pool of Silencer Select Pre-Designed Tbx1 SiRNA (Life Technology) (final concentration 50 nmol/L) in antibiotic-free medium using Lipofectamine RNAiMAX Reagent (Life Technology) according to the manufacturer's instructions. 48hrs after siRNA transfection, cells were collected and processed for further analysis.
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