siRNA / miRNA gene silencing Mouse - RAW264.7 Rheb

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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ON-TARGETplus Mouse Rheb (19744) siRNA - SMARTpool

Horizon Discovery Ltd.

Upstream tips	Protocol tips	Downstream tips
	RAW 264.7 cells, a murine macrophage cell line, were acquired from ATCC (Manassas, VA) and were maintained in Dulbecco ’ s Modifi ed Eagle ’ s medium (GIBCO BRL, Grand Island, NY) containing 20 m MHEPES (Fisher scientifi c, Atlanta, GA), 10% FBS (GIBCO BRL), 100 U/ml of penicillin and 100 μg/ml of streptomycin (Bio Whittaker Inc., Walkersville, MD) at 37 ° C in 5% CO 2. The cells were plated onto 6- or 96-well plates.For silencing Beclin 1, BNIP3, Rheb and Atg5 genes, Beclin 1-directed siRNA pool (ON-TARGET plus SMARTpool reagent L-055895-00-0005), BNIP3-directed siRNA pool (ON-TARGET plus SMARTpool reagent L-040256-01-0005) and Rhebdirected siRNA pool (ON-TARGET plus SMARTpool reagent L-057044-00-0005) were purchased from Dharmacon (Lafayette, CO). Atg5-directed siRNA was purchased from Ambion (Austin, TX). The sequence of Atg5 siRNA is 5 ’ -ACC GGA AAC TCA TGG AAT A-3 ’ . Negative control siRNA was purchased from Dharmacon. Cells were transfected with Beclin 1, BNIP3, Rheb, Atg5 or control siRNA by electroporation under conditions of 1350 voltage and 35 ms using a pipettetype electroporator (MicroPorator-Mini, Digital Bio Technology, Suwon, Kyounggi-do, Korea) according to the manufacturer ’ s instructions. The effi ciency of the siRNA silencing of the designated gene was confi rmed by western blot analysis at 48 h after transfection

Protocol tips
RAW 264.7 cells, a murine macrophage cell line, were acquired from ATCC (Manassas, VA) and were maintained in Dulbecco ’ s Modifi ed Eagle ’ s medium (GIBCO BRL, Grand Island, NY) containing 20 m MHEPES (Fisher scientifi c, Atlanta, GA), 10% FBS (GIBCO BRL), 100 U/ml of penicillin and 100 μg/ml of streptomycin (Bio Whittaker Inc., Walkersville, MD) at 37 ° C in 5% CO 2. The cells were plated onto 6- or 96-well plates.For silencing Beclin 1, BNIP3, Rheb and Atg5 genes, Beclin 1-directed siRNA pool (ON-TARGET plus SMARTpool reagent L-055895-00-0005), BNIP3-directed siRNA pool (ON-TARGET plus SMARTpool reagent L-040256-01-0005) and Rhebdirected siRNA pool (ON-TARGET plus SMARTpool reagent L-057044-00-0005) were purchased from Dharmacon (Lafayette, CO). Atg5-directed siRNA was purchased from Ambion (Austin, TX). The sequence of Atg5 siRNA is 5 ’ -ACC GGA AAC TCA TGG AAT A-3 ’ . Negative control siRNA was purchased from Dharmacon. Cells were transfected with Beclin 1, BNIP3, Rheb, Atg5 or control siRNA by electroporation under conditions of 1350 voltage and 35 ms using a pipettetype electroporator (MicroPorator-Mini, Digital Bio Technology, Suwon, Kyounggi-do, Korea) according to the manufacturer ’ s instructions. The effi ciency of the siRNA silencing of the designated gene was confi rmed by western blot analysis at 48 h after transfection

Protocol tips

RAW 264.7 cells, a murine macrophage cell line, were acquired from ATCC (Manassas, VA) and were maintained in Dulbecco ’ s Modifi ed Eagle ’ s medium (GIBCO BRL, Grand Island, NY) containing 20 m MHEPES (Fisher scientifi c, Atlanta, GA), 10% FBS (GIBCO BRL), 100 U/ml of penicillin and 100 μg/ml of streptomycin (Bio Whittaker Inc., Walkersville, MD) at 37 ° C in 5% CO 2. The cells were plated onto 6- or 96-well plates.For silencing Beclin 1, BNIP3, Rheb and Atg5 genes, Beclin 1-directed siRNA pool (ON-TARGET plus SMARTpool reagent L-055895-00-0005), BNIP3-directed siRNA pool (ON-TARGET plus SMARTpool reagent L-040256-01-0005) and Rhebdirected siRNA pool (ON-TARGET plus SMARTpool reagent L-057044-00-0005) were purchased from Dharmacon (Lafayette, CO). Atg5-directed siRNA was purchased from Ambion (Austin, TX). The sequence of Atg5 siRNA is 5 ’ -ACC GGA AAC TCA TGG AAT A-3 ’ . Negative control siRNA was purchased from Dharmacon. Cells were transfected with Beclin 1, BNIP3, Rheb, Atg5 or control siRNA by electroporation under conditions of 1350 voltage and 35 ms using a pipettetype electroporator (MicroPorator-Mini, Digital Bio Technology, Suwon, Kyounggi-do, Korea) according to the manufacturer ’ s instructions. The effi ciency of the siRNA silencing of the designated gene was confi rmed by western blot analysis at 48 h after transfection

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