Silencer® GAPDH siRNA (human)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Seed 6.0 × 10^5 cells	siRNA concentration-1–30 nM Add diluted siRNA to diluted Lipofectamine® RNAiMAX Reagent (1:1 ratio). Incubate for 5 minutes at room temperature and add to cells. Incubate cells for 22h at 37°C

Upstream tips
Seed 6.0 × 10^5 cells
Protocol tips
siRNA concentration-1–30 nM Add diluted siRNA to diluted Lipofectamine® RNAiMAX Reagent (1:1 ratio). Incubate for 5 minutes at room temperature and add to cells. Incubate cells for 22h at 37°C

Publication protocol

The evaluation of gene silencing of GAPDH was performed as reported previously15. Jurkat, THP-1, KG-1 or NK92 cells (6.0 × 105 cells) were seeded to 12 well plate and each carrier was added to the cells at siRNA doses of 1–30 nM. The RNAiMAX preparation was carried out following the manufacturer’s instructions. The RNAiMAX reagent (0.3 μl) was mixed with 1 nmol of siRNA. The cells were then incubated for 2 h at 37 °C in 0.5 ml of serum-free OPTI-MEM I. After a 2 h period of incubation, 0.5 ml of culture medium was added to the cells, followed by a further incubation for 22 h. After the incubation, the cells were collected and used for mRNA isolation using a RNeasy Mini Kit (QIAGEN, Hilden, Germany) according the manufacturer’s instructions. Briefly, the DNA contamination in the total RNA was eliminated by a DNase I treatment. The total RNA was then reverse-transcribed using a PrimeScript reverse transcription (RT) reagent Kit (Takara Bio Inc., Shiga, Japan) with oligo-dT primer. Quantitative polymerase chain reaction (PCR) was performed with a Mx3000 P QPCR System (Agilent Technologies, Santa Clara, CA) in 25 μl aliquots of reaction mixures containing cDNA, appropriate pairs of primers and THUNDERBIRD SYBR qPCR Mix (TOYOBO Co., Osaka, Japan). GAPDH level was calculated by the comparative CT method using beta actin as endogenous housekeeping genes. The following primer pairs were used: GAPDH: 5′-CCTCTGACTTCAACAGCGAC-3′ (forward); 5′-CGTTGTCATACCAGGAAATGAG-3′ (reverse); beta actin: 5′-CACTCTTCCAGCCTTCCTTC-3′ (forward); 5′-TACAGGTCTTTGCGCATGTC-3′ (reverse).

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Manufacturer protocol

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