siRNA / miRNA gene silencing Human - HCT-116 TET3(TET methylcytosine dioxygenase 3)

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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Found 1 matching solution for this experiment

Hs_TET3_2 FlexiTube siRNA

Qiagen

Upstream tips	Protocol tips	Downstream tips
	CT116 colorectal carcinoma cells were sourced from the American Type Culture Collection. HCT116 cells were cultured in Roswell Park Memorial Institute 1640 (RPMI-1640) medium supplemented with 10% fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc.), 50 U/ml penicillin/streptomycin and 2 g/l sodium bicarbonate. Transfection was done using Lipofectamin 2000. The siRNAs were designed to specifically target their corresponding transcripts. Designed with asymmetrical 5′base pair stabilities, these double-stranded oligos ensure entry of less tightly-bound antisense strand to the RNA-induced silencing complex while reducing risk of off-target effects by degrading the sense strand, which can be incorrectly processed by the RNA-induced silencing complex. The optimum concentration for each siRNA was 30 nM. RNA extraction with TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) of siRNA-trans-fected cells was performed according to the manufacturer's protocol, and the generated corresponding cDNA was used for RT-qPCR to validate the efficacy of the siRNAs.

Protocol tips
CT116 colorectal carcinoma cells were sourced from the American Type Culture Collection. HCT116 cells were cultured in Roswell Park Memorial Institute 1640 (RPMI-1640) medium supplemented with 10% fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc.), 50 U/ml penicillin/streptomycin and 2 g/l sodium bicarbonate. Transfection was done using Lipofectamin 2000. The siRNAs were designed to specifically target their corresponding transcripts. Designed with asymmetrical 5′base pair stabilities, these double-stranded oligos ensure entry of less tightly-bound antisense strand to the RNA-induced silencing complex while reducing risk of off-target effects by degrading the sense strand, which can be incorrectly processed by the RNA-induced silencing complex. The optimum concentration for each siRNA was 30 nM. RNA extraction with TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) of siRNA-trans-fected cells was performed according to the manufacturer's protocol, and the generated corresponding cDNA was used for RT-qPCR to validate the efficacy of the siRNAs.

Protocol tips

CT116 colorectal carcinoma cells were sourced from the American Type Culture Collection. HCT116 cells were cultured in Roswell Park Memorial Institute 1640 (RPMI-1640) medium supplemented with 10% fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc.), 50 U/ml penicillin/streptomycin and 2 g/l sodium bicarbonate. Transfection was done using Lipofectamin 2000. The siRNAs were designed to specifically target their corresponding transcripts. Designed with asymmetrical 5′base pair stabilities, these double-stranded oligos ensure entry of less tightly-bound antisense strand to the RNA-induced silencing complex while reducing risk of off-target effects by degrading the sense strand, which can be incorrectly processed by the RNA-induced silencing complex. The optimum concentration for each siRNA was 30 nM. RNA extraction with TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) of siRNA-trans-fected cells was performed according to the manufacturer's protocol, and the generated corresponding cDNA was used for RT-qPCR to validate the efficacy of the siRNAs.

Manufacturer protocol Publication protocol 1 Relevant paper

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