siRNA / miRNA gene silencing Rat - NRVM( Mapk14/p38

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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Silencer® Select_Mapk14/p38 siRNA(r)

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	Primary neonatal rat ventricular myocyte (NRVM) cultures were prepared from 1–2-day-old rats using standard methods as previously described [10]. Cells were cultured in growth medium consisting of 10% Horse Serum (HS), 5% Fetal Bovine Serum (FBS) and 1% Penicillin/Streptomycin (PS) in F-10 Nutrient Mixture Media. The experiments were carried 36 h after cells were seeded. Pre-designed siRNA targeting rat p38 mRNA and an siRNA control were obtained from Invitrogen (siRNA p38 s135447 and siRNA control AM4611). siRNA transfection was performed using Lipofectamine RNAiMAX according to the manufacturer's instructions (Invitrogen) with slight modifications. Briefly, 0.5×106 NRVMs were transfected in 2 ml of F-10 medium containing 500 μl of Opti-MEM (Invitrogen), 8 μl of Lipofectamine RNAiMAX and 100 nmol of siRNA.

Protocol tips
Primary neonatal rat ventricular myocyte (NRVM) cultures were prepared from 1–2-day-old rats using standard methods as previously described [10]. Cells were cultured in growth medium consisting of 10% Horse Serum (HS), 5% Fetal Bovine Serum (FBS) and 1% Penicillin/Streptomycin (PS) in F-10 Nutrient Mixture Media. The experiments were carried 36 h after cells were seeded. Pre-designed siRNA targeting rat p38 mRNA and an siRNA control were obtained from Invitrogen (siRNA p38 s135447 and siRNA control AM4611). siRNA transfection was performed using Lipofectamine RNAiMAX according to the manufacturer's instructions (Invitrogen) with slight modifications. Briefly, 0.5×106 NRVMs were transfected in 2 ml of F-10 medium containing 500 μl of Opti-MEM (Invitrogen), 8 μl of Lipofectamine RNAiMAX and 100 nmol of siRNA.

Protocol tips

Primary neonatal rat ventricular myocyte (NRVM) cultures were prepared from 1–2-day-old rats using standard methods as previously described [10]. Cells were cultured in growth medium consisting of 10% Horse Serum (HS), 5% Fetal Bovine Serum (FBS) and 1% Penicillin/Streptomycin (PS) in F-10 Nutrient Mixture Media. The experiments were carried 36 h after cells were seeded. Pre-designed siRNA targeting rat p38 mRNA and an siRNA control were obtained from Invitrogen (siRNA p38 s135447 and siRNA control AM4611). siRNA transfection was performed using Lipofectamine RNAiMAX according to the manufacturer's instructions (Invitrogen) with slight modifications. Briefly, 0.5×106 NRVMs were transfected in 2 ml of F-10 medium containing 500 μl of Opti-MEM (Invitrogen), 8 μl of Lipofectamine RNAiMAX and 100 nmol of siRNA.

Manufacturer protocol Publication protocol 1 Relevant paper

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