siRNA / miRNA gene silencing Human - A431 KRAS

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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KRAS siRNA

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	Human epidermoid carcinoma cell line A431 (DSMZ, Braunschweig, Germany) was kept in RPMI 1640 medium. Cell culture media were supplemented with 10% (vol/vol) heat-inactivated fetal calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were seeded at a density of 1 x 105/well in six-well plates. Next day, transfection of small interfering RNA (siRNA) was carried out using Lipofectamine2000 (Invitrogen, Carlsbad, CA). For all procedures, standard protocols were used according to the manufacturers' manuals. Synthetic siRNAs targeting KRAS4b was purchased from Applied Biosystems/Ambion (Carlsbad, CA). Target sequences were as follows: KRAS siRNA 1 (ID s7939) sense 5′-CUAUGGUCCUAGUAGGAAAtt-3′ and antisense 5′-UUUCCUACUAGGACCAUAGgt-3′; KRAS siRNA 2 (ID s7940) sense 5′-GCCUUGACGAUACAGCUAAtt-3′ and antisense 5′-UUAGCUGUAUCGUCAAGGCac-3′; As a control, unspecific siRNA Negative Control 1 (Applied Biosystems/Ambion) was used. Cells were transfected with 25 nM KRAS4b-specific or control siRNA for 72 hours.

Protocol tips
Human epidermoid carcinoma cell line A431 (DSMZ, Braunschweig, Germany) was kept in RPMI 1640 medium. Cell culture media were supplemented with 10% (vol/vol) heat-inactivated fetal calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were seeded at a density of 1 x 105/well in six-well plates. Next day, transfection of small interfering RNA (siRNA) was carried out using Lipofectamine2000 (Invitrogen, Carlsbad, CA). For all procedures, standard protocols were used according to the manufacturers' manuals. Synthetic siRNAs targeting KRAS4b was purchased from Applied Biosystems/Ambion (Carlsbad, CA). Target sequences were as follows: KRAS siRNA 1 (ID s7939) sense 5′-CUAUGGUCCUAGUAGGAAAtt-3′ and antisense 5′-UUUCCUACUAGGACCAUAGgt-3′; KRAS siRNA 2 (ID s7940) sense 5′-GCCUUGACGAUACAGCUAAtt-3′ and antisense 5′-UUAGCUGUAUCGUCAAGGCac-3′; As a control, unspecific siRNA Negative Control 1 (Applied Biosystems/Ambion) was used. Cells were transfected with 25 nM KRAS4b-specific or control siRNA for 72 hours.

Protocol tips

Human epidermoid carcinoma cell line A431 (DSMZ, Braunschweig, Germany) was kept in RPMI 1640 medium. Cell culture media were supplemented with 10% (vol/vol) heat-inactivated fetal calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were seeded at a density of 1 x 105/well in six-well plates. Next day, transfection of small interfering RNA (siRNA) was carried out using Lipofectamine2000 (Invitrogen, Carlsbad, CA). For all procedures, standard protocols were used according to the manufacturers' manuals. Synthetic siRNAs targeting KRAS4b was purchased from Applied Biosystems/Ambion (Carlsbad, CA). Target sequences were as follows: KRAS siRNA 1 (ID s7939) sense 5′-CUAUGGUCCUAGUAGGAAAtt-3′ and antisense 5′-UUUCCUACUAGGACCAUAGgt-3′; KRAS siRNA 2 (ID s7940) sense 5′-GCCUUGACGAUACAGCUAAtt-3′ and antisense 5′-UUAGCUGUAUCGUCAAGGCac-3′; As a control, unspecific siRNA Negative Control 1 (Applied Biosystems/Ambion) was used. Cells were transfected with 25 nM KRAS4b-specific or control siRNA for 72 hours.

Manufacturer protocol Publication protocol 1 Relevant paper

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