Cnp siRNA

siRNA / miRNA gene silencing Mouse - BV2 CNPase

Experiment
siRNA / miRNA gene silencing Mouse - BV2 CNPase
Product
Cnp siRNA from Thermo Fisher Scientific
Manufacturer
Thermo Fisher Scientific

Protocol tips

Upstream tips
Seed 3 × 10^5 cells/well.

Protocol tips
siRNA concentration 10 μM.

Incubate with the siRNA transfection mixture for 8 hours.

Replace medium and incubate for another 16 hours.

Publication protocol

Two constructs of mouse CNPase-specific siRNA (Ambion, Foster City, CA, USA; siRNA ID: s64160 & s64161; Cat. No. 4390771) were used for CNPase silencing. Nonspecific scramble siRNA (Ambion; Cat. No. 4390846) was used as control siRNA. To achieve a higher siRNA knockdown efficiency, the reverse transfection method was adopted for silencing according to the manufacturer’s instructions. Briefly, sub-confluent, early passage BV-2 cells were harvested by trypsinization, centrifuged and resuspended in Optimem (GIBCO, Invitrogen, Carlsbad, CA, USA; Cat. No. 31985070) and plated in 6-well plates at a density of ~3 × 105 cells/well. This was followed by adding 500 μl Optimem with 5 μl siRNA (10 μM) and 4 μl Lipofectamine® RNAiMAX Transfection Reagent (Invitrogen; Cat. No. 13778075) dropwise in the above well. The cells were incubated with the siRNA transfection mixture for 8 hours and then the medium was replaced with DMEM with 10% FBS without antibiotics and incubated for another 16 hours for RNA extraction to detect the knockdown efficiency by reverse transcription (RT)-PCR. Microglial cells were subjected to LPS treatment for 6 hours at 42 hours after transfection. After that, cells were either fixed for immunofluorescence staining, or mRNA and protein extracted for real-time RT-PCR and western blotting, respectively.

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Manufacturer protocol

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