siRNA / miRNA gene silencing Human - MDA-MB-453 GIRK1/KCNJ3

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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stealth siRNA GIRK1/KCNJ3

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	The human estrogen non-responsive MDA-MB-453 breast cancer cell line was purchased from the American Type Culture Collection (Rockville, MD, U.S.A.) and was maintained in RPMI 1640 medium supplemented with fetal bovine serum (10%, v/v), L-glutamine (2 mM), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C in an environment of 5% CO2.Three stealth siRNA constructs (Invitrogen, Carlsbad, CA, U.S.A.) were made starting at bases 1104, 1315, and 1490 of the GIRK1 sequence (GenBank # NM_002239). These constructs are: 1104 sense—gga aac aac ugg gau gac uug uca a; 1104 antisense—uug aca agu cau ccc agu ugu uuc c (GC content 44%); 1315 sense—cca gcc aua acu aac agc aaa gaa a; 1315 antisense—uuu cuu ugc ugu uag uua ugg cug g (GC content 40%); 1490 sense—acu ugc cca uga aac uuc aac gaa u; 1490 antisense—auu cgu uga agu uuc aug ggc aag u (GC content 40%). In addition, the low GC RNAi negative control (35%–45% GC) was used as recommended by the manufacturer (Invitrogen). These constructs (200 pMol) were transfected into the MDA-MB-453 breast cancer cell lines using lipofectamine 2000 (Invitrogen).

Protocol tips
The human estrogen non-responsive MDA-MB-453 breast cancer cell line was purchased from the American Type Culture Collection (Rockville, MD, U.S.A.) and was maintained in RPMI 1640 medium supplemented with fetal bovine serum (10%, v/v), L-glutamine (2 mM), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C in an environment of 5% CO2.Three stealth siRNA constructs (Invitrogen, Carlsbad, CA, U.S.A.) were made starting at bases 1104, 1315, and 1490 of the GIRK1 sequence (GenBank # NM_002239). These constructs are: 1104 sense—gga aac aac ugg gau gac uug uca a; 1104 antisense—uug aca agu cau ccc agu ugu uuc c (GC content 44%); 1315 sense—cca gcc aua acu aac agc aaa gaa a; 1315 antisense—uuu cuu ugc ugu uag uua ugg cug g (GC content 40%); 1490 sense—acu ugc cca uga aac uuc aac gaa u; 1490 antisense—auu cgu uga agu uuc aug ggc aag u (GC content 40%). In addition, the low GC RNAi negative control (35%–45% GC) was used as recommended by the manufacturer (Invitrogen). These constructs (200 pMol) were transfected into the MDA-MB-453 breast cancer cell lines using lipofectamine 2000 (Invitrogen).

Protocol tips

The human estrogen non-responsive MDA-MB-453 breast cancer cell line was purchased from the American Type Culture Collection (Rockville, MD, U.S.A.) and was maintained in RPMI 1640 medium supplemented with fetal bovine serum (10%, v/v), L-glutamine (2 mM), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C in an environment of 5% CO2.Three stealth siRNA constructs (Invitrogen, Carlsbad, CA, U.S.A.) were made starting at bases 1104, 1315, and 1490 of the GIRK1 sequence (GenBank # NM_002239). These constructs are: 1104 sense—gga aac aac ugg gau gac uug uca a; 1104 antisense—uug aca agu cau ccc agu ugu uuc c (GC content 44%); 1315 sense—cca gcc aua acu aac agc aaa gaa a; 1315 antisense—uuu cuu ugc ugu uag uua ugg cug g (GC content 40%); 1490 sense—acu ugc cca uga aac uuc aac gaa u; 1490 antisense—auu cgu uga agu uuc aug ggc aag u (GC content 40%). In addition, the low GC RNAi negative control (35%–45% GC) was used as recommended by the manufacturer (Invitrogen). These constructs (200 pMol) were transfected into the MDA-MB-453 breast cancer cell lines using lipofectamine 2000 (Invitrogen).

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