siRNA / miRNA gene silencing Rat - PC12 Atf4

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.

1 Matching solution
Start a discussion

Start discussion

No discussions found

Start your discussion

Share your thoughts or question with experts in your field

Start a discussion

Found 1 matching solution for this experiment

ON-TARGETplus Rat Atf4 (79255) siRNA - SMARTpool

Horizon Discovery Ltd.

Upstream tips	Protocol tips	Downstream tips
	Gene silencing was performed using siRNAs. GRP78-specific siRNAs were synthesized by Shanghai GenePharma (Shanghai, China) with the sense strand sequence of 5′-GAGGCGUAUUUGGGAAAGATT-3′.The scrambled siRNA has the sense sequence of 5′-UUCUCCGAACGUGUCACGUTT-3′. To efficiently knock down ATF4, we used a pool of four different siRNAs (ON-TARGET plus SMART pool Rat ATF4; Thermo Scientific Dharmacon) to target rat ATF4 mRNA. Control cultures were incubated with non-targeting siRNAs (ON-TARGET plus non-targeting siRNA#1; Thermo Scientific Dharmacon).Transfection of siRNAs was carried out using Lipofectamine™ 2000 (Invitrogen, Grand Island, NY, USA), according to the manufacturer's instructions. Briefly, siRNA and Lipofectamine™ 2000 reagent were mixed in Opti-MEM medium (Invitrogen) and incubated for 30 min at room temperature to allow the complex formation. Cells were washed with Opti-MEM medium, and the transfection mixture was added. 6 h after transfection, cells were washed and cultured for 24 h in complete medium containing 10% FCS and 5% FBS. The silencing efficacy was evaluated by western blotting.

Protocol tips
Gene silencing was performed using siRNAs. GRP78-specific siRNAs were synthesized by Shanghai GenePharma (Shanghai, China) with the sense strand sequence of 5′-GAGGCGUAUUUGGGAAAGATT-3′.The scrambled siRNA has the sense sequence of 5′-UUCUCCGAACGUGUCACGUTT-3′. To efficiently knock down ATF4, we used a pool of four different siRNAs (ON-TARGET plus SMART pool Rat ATF4; Thermo Scientific Dharmacon) to target rat ATF4 mRNA. Control cultures were incubated with non-targeting siRNAs (ON-TARGET plus non-targeting siRNA#1; Thermo Scientific Dharmacon).Transfection of siRNAs was carried out using Lipofectamine™ 2000 (Invitrogen, Grand Island, NY, USA), according to the manufacturer's instructions. Briefly, siRNA and Lipofectamine™ 2000 reagent were mixed in Opti-MEM medium (Invitrogen) and incubated for 30 min at room temperature to allow the complex formation. Cells were washed with Opti-MEM medium, and the transfection mixture was added. 6 h after transfection, cells were washed and cultured for 24 h in complete medium containing 10% FCS and 5% FBS. The silencing efficacy was evaluated by western blotting.

Protocol tips

Gene silencing was performed using siRNAs. GRP78-specific siRNAs were synthesized by Shanghai GenePharma (Shanghai, China) with the sense strand sequence of 5′-GAGGCGUAUUUGGGAAAGATT-3′.The scrambled siRNA has the sense sequence of 5′-UUCUCCGAACGUGUCACGUTT-3′. To efficiently knock down ATF4, we used a pool of four different siRNAs (ON-TARGET plus SMART pool Rat ATF4; Thermo Scientific Dharmacon) to target rat ATF4 mRNA. Control cultures were incubated with non-targeting siRNAs (ON-TARGET plus non-targeting siRNA#1; Thermo Scientific Dharmacon).Transfection of siRNAs was carried out using Lipofectamine™ 2000 (Invitrogen, Grand Island, NY, USA), according to the manufacturer's instructions. Briefly, siRNA and Lipofectamine™ 2000 reagent were mixed in Opti-MEM medium (Invitrogen) and incubated for 30 min at room temperature to allow the complex formation. Cells were washed with Opti-MEM medium, and the transfection mixture was added. 6 h after transfection, cells were washed and cultured for 24 h in complete medium containing 10% FCS and 5% FBS. The silencing efficacy was evaluated by western blotting.

Manufacturer protocol Publication protocol 1 Relevant paper

Can't find the product you've used to perform this experiment? It would be great if you can help us by Adding a product!

Outsource your experiment

Fill out your contact details and receive price quotes in your Inbox

Outsource experiment