stealth siRNA GIRK1/KCNJ3

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	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).

Publication protocol

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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Manufacturer protocol

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