siRNA / miRNA gene silencing Human - HUVEC ADAMTS-13

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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ADAMTS13 siRNA

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	Human umbilical vein endothelial cells (HUVEC) were isolated from human umbilical veins as previously described (Tao et al., 2005) and cultured in M199 media supplemented with 20% fetal bovine serum penicillin, streptomycin, and amphotericin (Fungizone™). Upon reaching confluence, primary cultures of HUVEC were passaged using 0.25% trypsin/EDTA. Second-passage HUVEC were utilized in all assays. 24 h prior to the start of the knockdown assay, 2 × 105 first-passage HUVEC were added to a six-well plate in 2 ml of M199 media containing 20% FBS with antibiotics, such that approximately 60–80% confluence would be reached by the time of assay. The control and ADAMTS-13 siRNA knockdown assays were carried out according to the manufacturer's protocol. The cells or the cell lysates were harvested after 24 h incubation. ADAMTS-13 protein level in cell lysates from HUVEC treated with control siRNA or three different ADAMTS-13 siRNAs (s21868, s21869 and s21870). 15 nM siRNA was used for each transfection. After 24 h, cell lysates were harvested, concentrated and assayed with an ADAMTS-13 ELISA kit.

Protocol tips
Human umbilical vein endothelial cells (HUVEC) were isolated from human umbilical veins as previously described (Tao et al., 2005) and cultured in M199 media supplemented with 20% fetal bovine serum penicillin, streptomycin, and amphotericin (Fungizone™). Upon reaching confluence, primary cultures of HUVEC were passaged using 0.25% trypsin/EDTA. Second-passage HUVEC were utilized in all assays. 24 h prior to the start of the knockdown assay, 2 × 105 first-passage HUVEC were added to a six-well plate in 2 ml of M199 media containing 20% FBS with antibiotics, such that approximately 60–80% confluence would be reached by the time of assay. The control and ADAMTS-13 siRNA knockdown assays were carried out according to the manufacturer's protocol. The cells or the cell lysates were harvested after 24 h incubation. ADAMTS-13 protein level in cell lysates from HUVEC treated with control siRNA or three different ADAMTS-13 siRNAs (s21868, s21869 and s21870). 15 nM siRNA was used for each transfection. After 24 h, cell lysates were harvested, concentrated and assayed with an ADAMTS-13 ELISA kit.

Protocol tips

Human umbilical vein endothelial cells (HUVEC) were isolated from human umbilical veins as previously described (Tao et al., 2005) and cultured in M199 media supplemented with 20% fetal bovine serum penicillin, streptomycin, and amphotericin (Fungizone™). Upon reaching confluence, primary cultures of HUVEC were passaged using 0.25% trypsin/EDTA. Second-passage HUVEC were utilized in all assays. 24 h prior to the start of the knockdown assay, 2 × 105 first-passage HUVEC were added to a six-well plate in 2 ml of M199 media containing 20% FBS with antibiotics, such that approximately 60–80% confluence would be reached by the time of assay. The control and ADAMTS-13 siRNA knockdown assays were carried out according to the manufacturer's protocol. The cells or the cell lysates were harvested after 24 h incubation. ADAMTS-13 protein level in cell lysates from HUVEC treated with control siRNA or three different ADAMTS-13 siRNAs (s21868, s21869 and s21870). 15 nM siRNA was used for each transfection. After 24 h, cell lysates were harvested, concentrated and assayed with an ADAMTS-13 ELISA kit.

Manufacturer protocol Publication protocol 1 Relevant paper

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