siRNA / miRNA gene silencing Mouse - Neuro 2a Fpr1/Fpr2

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_FPr1/Silencer® Select_FPr2 siRNA

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	Mouse Neuroblastoma cells (N2a (ECACC 89121404) were obtained from European Collection of Authenticated Cell Cultures (UK). Eagle’s Minimum Essential Medium high glucose (EMEM), Dulbecco’s Modified Eagle’s Medium high glucose (DMEM), all SilencerTM select siRNA duplexes for mouse Fpr1 (siRNA ID s66215), mouse Fpr2 (siRNA ID 66212) plus negative control no.1 siRNA and Lipofectamine RNAiMAX were purchased from ThermoFisher Scientific (UK). All other chemicals used were of reagent grade. N2a cells were cultured in DMEM containing 2mM glutamine, 100μg/ml penicillin, 100μg/ml streptomycin, and 10% (v/v) heat inactivated fetal bovine serum (complete DMEM). IMR-32 and SH-SY5Y cells were cultured in EMEM containing 2mM glutamine, 100μg/ml penicillin, 100μg/ml streptomycin, and 10% (v/v) heat inactivated fetal bovine serum (complete EMEM). For siRNA transfections N2a cells were cultured in serum-free antibiotic-free DMEM. Cells were incubated under standard conditions of: 37°C, 5% CO2 in a humidified atmosphere. Cultures were passaged at regular intervals, once at 70–80% confluence.Two siRN A duplexes targeting Fpr1, Fpr2 plus a third negative control knockdown duplex were used. Transfections were carried out using Lipofectamine RNAiMAX in antibiotic-free serum-free DMEM. Lipofectamine RNAiMAX and siRNA duplexes (10μM stock in nuclease-free water) were diluted separately in antibiotic-free serum-free DMEM in a v/v ratio of 1.5:25 and 1:50 respectively. Diluted siRNA was then added to diluted Lipofectamine in a 1:1 ratio and incubated at room temperature for 5min. siRNA-lipid complexes then added to sub-confluent N2a cells; 10μL per well for 96-well plate assays giving a final siRNA concentration of 1pM and 0.3μL Lipofectamine per well, and 50μL per well was used for 24-well plate assays giving a final siRNA concentration of 5pM and 1.5μL Lipofectamine per well. For simultaneous Fpr1 and Fpr2 knockdown, the Fpr1 and Fpr2 siRNA duplexes were added to 0.6μL Lipofectamine (96-well) at a final well concentration of 1pM, and 5pM with 3μL Lipofectamine (24-Well). Transfected cells were then incubated at 37°C, 5% CO2 for 48h before treatment with FPRa14 agonist [28].

Protocol tips
Mouse Neuroblastoma cells (N2a (ECACC 89121404) were obtained from European Collection of Authenticated Cell Cultures (UK). Eagle’s Minimum Essential Medium high glucose (EMEM), Dulbecco’s Modified Eagle’s Medium high glucose (DMEM), all SilencerTM select siRNA duplexes for mouse Fpr1 (siRNA ID s66215), mouse Fpr2 (siRNA ID 66212) plus negative control no.1 siRNA and Lipofectamine RNAiMAX were purchased from ThermoFisher Scientific (UK). All other chemicals used were of reagent grade. N2a cells were cultured in DMEM containing 2mM glutamine, 100μg/ml penicillin, 100μg/ml streptomycin, and 10% (v/v) heat inactivated fetal bovine serum (complete DMEM). IMR-32 and SH-SY5Y cells were cultured in EMEM containing 2mM glutamine, 100μg/ml penicillin, 100μg/ml streptomycin, and 10% (v/v) heat inactivated fetal bovine serum (complete EMEM). For siRNA transfections N2a cells were cultured in serum-free antibiotic-free DMEM. Cells were incubated under standard conditions of: 37°C, 5% CO2 in a humidified atmosphere. Cultures were passaged at regular intervals, once at 70–80% confluence.Two siRN A duplexes targeting Fpr1, Fpr2 plus a third negative control knockdown duplex were used. Transfections were carried out using Lipofectamine RNAiMAX in antibiotic-free serum-free DMEM. Lipofectamine RNAiMAX and siRNA duplexes (10μM stock in nuclease-free water) were diluted separately in antibiotic-free serum-free DMEM in a v/v ratio of 1.5:25 and 1:50 respectively. Diluted siRNA was then added to diluted Lipofectamine in a 1:1 ratio and incubated at room temperature for 5min. siRNA-lipid complexes then added to sub-confluent N2a cells; 10μL per well for 96-well plate assays giving a final siRNA concentration of 1pM and 0.3μL Lipofectamine per well, and 50μL per well was used for 24-well plate assays giving a final siRNA concentration of 5pM and 1.5μL Lipofectamine per well. For simultaneous Fpr1 and Fpr2 knockdown, the Fpr1 and Fpr2 siRNA duplexes were added to 0.6μL Lipofectamine (96-well) at a final well concentration of 1pM, and 5pM with 3μL Lipofectamine (24-Well). Transfected cells were then incubated at 37°C, 5% CO2 for 48h before treatment with FPRa14 agonist [28].

Protocol tips

Mouse Neuroblastoma cells (N2a (ECACC 89121404) were obtained from European Collection of Authenticated Cell Cultures (UK). Eagle’s Minimum Essential Medium high glucose (EMEM), Dulbecco’s Modified Eagle’s Medium high glucose (DMEM), all SilencerTM select siRNA duplexes for mouse Fpr1 (siRNA ID s66215), mouse Fpr2 (siRNA ID 66212) plus negative control no.1 siRNA and Lipofectamine RNAiMAX were purchased from ThermoFisher Scientific (UK). All other chemicals used were of reagent grade. N2a cells were cultured in DMEM containing 2mM glutamine, 100μg/ml penicillin, 100μg/ml streptomycin, and 10% (v/v) heat inactivated fetal bovine serum (complete DMEM). IMR-32 and SH-SY5Y cells were cultured in EMEM containing 2mM glutamine, 100μg/ml penicillin, 100μg/ml streptomycin, and 10% (v/v) heat inactivated fetal bovine serum (complete EMEM). For siRNA transfections N2a cells were cultured in serum-free antibiotic-free DMEM. Cells were incubated under standard conditions of: 37°C, 5% CO2 in a humidified atmosphere. Cultures were passaged at regular intervals, once at 70–80% confluence.Two siRN A duplexes targeting Fpr1, Fpr2 plus a third negative control knockdown duplex were used. Transfections were carried out using Lipofectamine RNAiMAX in antibiotic-free serum-free DMEM. Lipofectamine RNAiMAX and siRNA duplexes (10μM stock in nuclease-free water) were diluted separately in antibiotic-free serum-free DMEM in a v/v ratio of 1.5:25 and 1:50 respectively. Diluted siRNA was then added to diluted Lipofectamine in a 1:1 ratio and incubated at room temperature for 5min. siRNA-lipid complexes then added to sub-confluent N2a cells; 10μL per well for 96-well plate assays giving a final siRNA concentration of 1pM and 0.3μL Lipofectamine per well, and 50μL per well was used for 24-well plate assays giving a final siRNA concentration of 5pM and 1.5μL Lipofectamine per well. For simultaneous Fpr1 and Fpr2 knockdown, the Fpr1 and Fpr2 siRNA duplexes were added to 0.6μL Lipofectamine (96-well) at a final well concentration of 1pM, and 5pM with 3μL Lipofectamine (24-Well). Transfected cells were then incubated at 37°C, 5% CO2 for 48h before treatment with FPRa14 agonist [28].

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