A siRNA library containing 3 siRNAs per kinase for 710 human kinase genes was purchased from Ambion (catalog #4397918). The siRNAs in the 96-well plates were resuspended in nuclease-free sterile water to a concentration of 1 μM. Aliquots of the siRNAs were further diluted into daughter plates at a concentration of 250 nM. Daoy cells from the same passage were used to perform the screen. Briefly, Daoy cells were reverse transfected using siPORT NeoFX Transfection Agent with a final siRNA concentration of 5 nM. A separate triplicate set of wells containing cells transfected with a non-silencing siRNA were included on each plate. A MTS assay was performed 72 hours after transfection to determine whether the particular gene targeted by the siRNA had any effect on cell proliferation.
Sirna library
Manufactured by Thermo Fisher Scientific
The SiRNA library is a collection of small interfering RNA (siRNA) molecules designed to target and silence specific genes. The core function of this product is to provide researchers with a tool for studying gene function and knockdown in various biological systems.
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Lab products found in correlation
2 protocols using sirna library
1
Kinase-Targeted siRNA Library Screening
2
Silencing ATP7A Gene Expression
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ATP7A siRNA sequences were obtained from Ambion (Austin, TX) siRNA library (ID number 120175) and targeted exon 11 (sense, GCAACUAUUGUAACUCUUG dTdT; antisense, CAAGAGUUACAAUAGUUGC dTdT) [30]. A nonsilencing siRNA sequence, shown by BLAST search to not share sequence homology with any known human mRNA was used as control for ATP7A-targeting experiments. The siRNA sequences were synthesized by Guangzhou RiboBio (Ghuangzhou, China). EC109/DDP or A549/DDP cells were plated into 6-well plates as required for the experiments. The cells were allowed to adhere for overnight. The transfection of siRNA was performed using lipofectamine-2000 (Invitrogen, CA, USA) according to the manufacturer's recommendation. Oligofectamine /siRNA complexes were formed in serum free DMEM by adding siRNA (25, 50 or 100 nM final concentration) to 5μl of Oligofectamine (Invitrogen, CA, USA) per well. Complexes were allowed to form at 25°C for 10 min and added to wells (500 μl per well). After 4 hours of transfection, the culture medium containing 10% serum was added. The assays were carried out 24, 48, 72, 80 hours post transfection.
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