siRNA / miRNA gene silencing Human - HeLa ARAP1

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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Found 1 matching solution for this experiment

siGENOME Human ARAP1 (116985) siRNA - SMARTpool

Horizon Discovery Ltd.

Upstream tips	Protocol tips	Downstream tips
	siRNA oligonucleotides were purchased from Dharmacon [catalogue number M‐009136‐02 (Smart pool) and MU‐009136‐02 (set of four Upgrade siRNA duplexes) for human ARAP1, LOC361617 for rat ARAP1 and D‐001810‐10 for ON‐TARGETplus siCONTROL non‐targeting pool] and transfected using Oligofectamine (Invitrogen), according to the manufacturer’s instructions. The four oligonucleotides present in the pool that were also tested separately were 1 (5′‐GAGAAGGAGUGGCCUAUUA‐3′), 2 (5′‐GCAGGGAUCUUACAUCUAU‐3′), 3 (5′‐GGAGAUCACUGCCAUUGUG‐3′) and 4 (5′‐ACAAGAAUCUAGAGGAGUA‐3′).A significant reduction in ARAP1 levels was obtained after 72 h siRNAs treatment in HeLa cells (80 ± 5%) and after 48 h in NRK cells (70 ± 7%). All the experiments reported were performed in HeLa cells using the Smart pool of the four oligonucleotides and then repeated with at least two different single oligonucleotides (mainly 1 and 4).

Protocol tips
siRNA oligonucleotides were purchased from Dharmacon [catalogue number M‐009136‐02 (Smart pool) and MU‐009136‐02 (set of four Upgrade siRNA duplexes) for human ARAP1, LOC361617 for rat ARAP1 and D‐001810‐10 for ON‐TARGETplus siCONTROL non‐targeting pool] and transfected using Oligofectamine (Invitrogen), according to the manufacturer’s instructions. The four oligonucleotides present in the pool that were also tested separately were 1 (5′‐GAGAAGGAGUGGCCUAUUA‐3′), 2 (5′‐GCAGGGAUCUUACAUCUAU‐3′), 3 (5′‐GGAGAUCACUGCCAUUGUG‐3′) and 4 (5′‐ACAAGAAUCUAGAGGAGUA‐3′).A significant reduction in ARAP1 levels was obtained after 72 h siRNAs treatment in HeLa cells (80 ± 5%) and after 48 h in NRK cells (70 ± 7%). All the experiments reported were performed in HeLa cells using the Smart pool of the four oligonucleotides and then repeated with at least two different single oligonucleotides (mainly 1 and 4).

Protocol tips

siRNA oligonucleotides were purchased from Dharmacon [catalogue number M‐009136‐02 (Smart pool) and MU‐009136‐02 (set of four Upgrade siRNA duplexes) for human ARAP1, LOC361617 for rat ARAP1 and D‐001810‐10 for ON‐TARGETplus siCONTROL non‐targeting pool] and transfected using Oligofectamine (Invitrogen), according to the manufacturer’s instructions. The four oligonucleotides present in the pool that were also tested separately were 1 (5′‐GAGAAGGAGUGGCCUAUUA‐3′), 2 (5′‐GCAGGGAUCUUACAUCUAU‐3′), 3 (5′‐GGAGAUCACUGCCAUUGUG‐3′) and 4 (5′‐ACAAGAAUCUAGAGGAGUA‐3′).A significant reduction in ARAP1 levels was obtained after 72 h siRNAs treatment in HeLa cells (80 ± 5%) and after 48 h in NRK cells (70 ± 7%). All the experiments reported were performed in HeLa cells using the Smart pool of the four oligonucleotides and then repeated with at least two different single oligonucleotides (mainly 1 and 4).

Manufacturer protocol Publication protocol 1 Relevant paper

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