siRNA / miRNA gene silencing Human - A375 CDK5RAP1

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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CDK5RAP1 siRNA (h)

Santa Cruz Biotechnology

Upstream tips	Protocol tips	Downstream tips
	The human malignant melanoma A375 cell line (A375-P) was purchased from American Type Culture Collection (Manassas, VA, USA). In accordance with experimental guidelines and ethical approval of Harbin Medical University (Harbin, China), the study was performed in Harbin Medical University. A375 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin (all from Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) with 5% CO2 at 37°C in a humidified incubator (Sanyo Electric Co., Ltd., Tokyo, Japan).Control siRNA and the CDK5RAP1 siRNA were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). A375 cells were seeded at a density of 1×105 cells/well onto six-well plates. After cells obtained 60–80% confluency, siRNAs were transfected into A375 cells according to the manufacturer's protocol. A375 cells were incubated for another 48 h prior to use in subsequent experiments. The transfection efficiency was analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), according to the protocol outlined below.

Protocol tips
The human malignant melanoma A375 cell line (A375-P) was purchased from American Type Culture Collection (Manassas, VA, USA). In accordance with experimental guidelines and ethical approval of Harbin Medical University (Harbin, China), the study was performed in Harbin Medical University. A375 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin (all from Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) with 5% CO2 at 37°C in a humidified incubator (Sanyo Electric Co., Ltd., Tokyo, Japan).Control siRNA and the CDK5RAP1 siRNA were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). A375 cells were seeded at a density of 1×105 cells/well onto six-well plates. After cells obtained 60–80% confluency, siRNAs were transfected into A375 cells according to the manufacturer's protocol. A375 cells were incubated for another 48 h prior to use in subsequent experiments. The transfection efficiency was analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), according to the protocol outlined below.

Protocol tips

The human malignant melanoma A375 cell line (A375-P) was purchased from American Type Culture Collection (Manassas, VA, USA). In accordance with experimental guidelines and ethical approval of Harbin Medical University (Harbin, China), the study was performed in Harbin Medical University. A375 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin (all from Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) with 5% CO2 at 37°C in a humidified incubator (Sanyo Electric Co., Ltd., Tokyo, Japan).Control siRNA and the CDK5RAP1 siRNA were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). A375 cells were seeded at a density of 1×105 cells/well onto six-well plates. After cells obtained 60–80% confluency, siRNAs were transfected into A375 cells according to the manufacturer's protocol. A375 cells were incubated for another 48 h prior to use in subsequent experiments. The transfection efficiency was analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), according to the protocol outlined below.

Manufacturer protocol Publication protocol 1 Relevant paper

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