siRNA / miRNA gene silencing Mouse - mESC Rbl2/p130

miRNA is the inherent gene silencing machinery which can have more than one mRNA target, whereas siRNA can be designed to target a particular mRNA target. By design, both siRNA and miRNA are 20-25 nucleotides in length. The target sequence for siRNAs is usually located within the open reading frame, between 50 and 100 nucleotides downstream of the start codon. There are two ways in which cells can be transfected with desired RNAi: 1. Direct transfection (with calcium phosphate co-precipitation or cationic lipid mediated transfection using lipofectamine or oligofectamine), and 2. Making RNAi lentiviral constructs (followed by transformation and transduction). Lentiviral constructs are time consuming, but provide a more permanent expression of RNAi in the cells, and consistent gene silencing. Direct transfection of oligonucleotides provides temporary genetic suppression. Traditional methods like calcium phosphate co-precipitation have challenges like low efficiency, poor reproducibility and cell toxicity. Whereas, cationic lipid-based transfection reagents are able to overcome these challenges, along with applicability to a large variety of eukaryotic cell lines. When using oligos, the ideal concentration lies between 10-50nM for effective transfection.

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p130 siRNA (m)

Santa Cruz Biotechnology

Upstream tips	Protocol tips	Downstream tips
	The D3 mESCs were cultured on a feeder layer of mitomycin-treated MEFs in a traditional complete medium (KS/LIF medium) containing knockout Dulbecco’s modified Eagle’s medium (DMEM/F-12) supplemented with 15% knockout serum replacement, 0.1 mM non-essential amino acids, 1% penicillin/streptomycin, 1 mM L-glutamine, 0.1 mM β-mercaptoethanol (Invitrogen) and 1000 U/ml mouse LIF (Millipore). 2i/LIF condition medium was made from the traditional complete medium by using 3 μM CHIR99021 (Stemgent) and 1 μM PD0325901 (Stemgent) to substitute knockout serum replacement. Prior to experiments, cells were passaged by incubation in 0.25% trypsin/EDTA for 5 min at 37 °C and then plated onto gelatin-coated dishes without a feeder layer for 24 h. Icaritin was purchased from Sigma. All reagents were purchased from Invitrogen unless otherwise specified.mESCs were transiently mESCs were transiently transfected with siRNAs specific for knockdown of Rbl2/p130, Cdx2 or ERα gene (Santa Cruz). Cells were plated on gelatin coated plates for 24 h. Transfection was preceded by using lipofectamine RNAiMAX reagent according to manufacturer’s instructions (Invitrogen). Control siRNA transfection was performed in parallel. Total RNA and protein was extracted at 48 h after transfection.with siRNAs specific for knockdown of Rbl2/p130, Cdx2 or ERα gene (Santa Cruz). Cells were plated on gelatin coated plates for 24 h. Transfection was preceded by using lipofectamine RNAiMAX reagent according to manufacturer’s instructions (Invitrogen). Control siRNA transfection was performed in parallel. Total RNA and protein was extracted at 48 h after transfection.

Protocol tips
The D3 mESCs were cultured on a feeder layer of mitomycin-treated MEFs in a traditional complete medium (KS/LIF medium) containing knockout Dulbecco’s modified Eagle’s medium (DMEM/F-12) supplemented with 15% knockout serum replacement, 0.1 mM non-essential amino acids, 1% penicillin/streptomycin, 1 mM L-glutamine, 0.1 mM β-mercaptoethanol (Invitrogen) and 1000 U/ml mouse LIF (Millipore). 2i/LIF condition medium was made from the traditional complete medium by using 3 μM CHIR99021 (Stemgent) and 1 μM PD0325901 (Stemgent) to substitute knockout serum replacement. Prior to experiments, cells were passaged by incubation in 0.25% trypsin/EDTA for 5 min at 37 °C and then plated onto gelatin-coated dishes without a feeder layer for 24 h. Icaritin was purchased from Sigma. All reagents were purchased from Invitrogen unless otherwise specified.mESCs were transiently mESCs were transiently transfected with siRNAs specific for knockdown of Rbl2/p130, Cdx2 or ERα gene (Santa Cruz). Cells were plated on gelatin coated plates for 24 h. Transfection was preceded by using lipofectamine RNAiMAX reagent according to manufacturer’s instructions (Invitrogen). Control siRNA transfection was performed in parallel. Total RNA and protein was extracted at 48 h after transfection.with siRNAs specific for knockdown of Rbl2/p130, Cdx2 or ERα gene (Santa Cruz). Cells were plated on gelatin coated plates for 24 h. Transfection was preceded by using lipofectamine RNAiMAX reagent according to manufacturer’s instructions (Invitrogen). Control siRNA transfection was performed in parallel. Total RNA and protein was extracted at 48 h after transfection.

Protocol tips

The D3 mESCs were cultured on a feeder layer of mitomycin-treated MEFs in a traditional complete medium (KS/LIF medium) containing knockout Dulbecco’s modified Eagle’s medium (DMEM/F-12) supplemented with 15% knockout serum replacement, 0.1 mM non-essential amino acids, 1% penicillin/streptomycin, 1 mM L-glutamine, 0.1 mM β-mercaptoethanol (Invitrogen) and 1000 U/ml mouse LIF (Millipore). 2i/LIF condition medium was made from the traditional complete medium by using 3 μM CHIR99021 (Stemgent) and 1 μM PD0325901 (Stemgent) to substitute knockout serum replacement. Prior to experiments, cells were passaged by incubation in 0.25% trypsin/EDTA for 5 min at 37 °C and then plated onto gelatin-coated dishes without a feeder layer for 24 h. Icaritin was purchased from Sigma. All reagents were purchased from Invitrogen unless otherwise specified.mESCs were transiently mESCs were transiently transfected with siRNAs specific for knockdown of Rbl2/p130, Cdx2 or ERα gene (Santa Cruz). Cells were plated on gelatin coated plates for 24 h. Transfection was preceded by using lipofectamine RNAiMAX reagent according to manufacturer’s instructions (Invitrogen). Control siRNA transfection was performed in parallel. Total RNA and protein was extracted at 48 h after transfection.with siRNAs specific for knockdown of Rbl2/p130, Cdx2 or ERα gene (Santa Cruz). Cells were plated on gelatin coated plates for 24 h. Transfection was preceded by using lipofectamine RNAiMAX reagent according to manufacturer’s instructions (Invitrogen). Control siRNA transfection was performed in parallel. Total RNA and protein was extracted at 48 h after transfection.

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