Hs_HDAC1_6 FlexiTube siRNA

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	siRNA concentration-40 nM Incubate the diluted oligonucleotide with diluted Oligofectamine   mixture for 15-20 minutes at room temperature. Mix well and incubate the cells at 37°C in a CO2 incubator for 4 hours and add growth medium containing 3X the normal concentration of serum without removing the transfection mixture. Incubate cells for 72 h at 37°C in a CO2 incubator

Protocol tips
siRNA concentration-40 nM Incubate the diluted oligonucleotide with diluted Oligofectamine   mixture for 15-20 minutes at room temperature. Mix well and incubate the cells at 37°C in a CO2 incubator for 4 hours and add growth medium containing 3X the normal concentration of serum without removing the transfection mixture. Incubate cells for 72 h at 37°C in a CO2 incubator

Publication protocol

A custom designed DUB siRNA library consisting of pools of four oligos for each of 92 human DUBs (Qiagen) and control siRNAs were previously used to prepare a library of fractionated protein extracts from A549 cells [14]. To screen for the effect of each DUB on HDAC levels, samples from this nuclear extract library were divided across four 4%–12% gradient gels that were run and processed in parallel. Following immunoblotting, the HDACs were imaged in separate channels and bands were quantified using the Odyssey system; the amount of HDAC2 was expressed relative to the amount of HDAC1. The value for each sample was then normalized to the median value for the corresponding immunoblot. Mean values were derived from two independent immunoblotting runs, before collation and ranking of the dataset. The individual siRNA sequences from each pool were used for initial validation. Two of these siRNAs hs_BAP1_3 (SI00066710) and hs_BAP1_5 (SI03036390) were used for follow-up studies together with hs_HDAC1_6 (SI02663472) and hs_HDAC2_1 (SI00434952) (Qiagen). Non-targeting control siRNAs used were siC (AllStars, Qiagen), and siCON1 (D-001210–01) or siCON2 (D-001210–02) from Dharmacon. For immunoblotting experiments, cells were seeded at 6×104 (A549 and NCI-H460) or 1.5×105 (MSTO-211H and COR-L23) cells per well in 6-well plates, respectively, transfected the following day with 40 nM siRNA using Oligofectamine (Invitrogen), and harvested after 72 hr

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Manufacturer protocol

Download the product protocol from Qiagen for Hs_HDAC1_6 FlexiTube siRNA below.

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