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Preparation of AteloGene® Local Use “Quick Gelation”
In this section, the numbers ① – ④ refer to the “Kit contents” on product page. Implement measures to secure an RNase-free environment, as far as possible, to avoid the degradation of siRNA/miRNA in advance.
1）Preparation of AteloGene® Local Use “Quick Gelation” (QG)
Attach the ④18G needle to the ①Prefilled syringe. Eject the whole amount (540 μL) into the ③2 mL microtube. After ejection, cool the microtube containing AteloGene® QG on ice.
An excess amount of AteloGene® QG is provided in the ①Prefilled syringe so that 540 μL of AteloGene® QG can be injected into the ③2.0 mL microtube regardless of losses during the preparation steps.
2） Preparation of siRNA/miRNA solution
Prepare 25 – 50 μM siRNA/miRNA solutions with RNase-free water.
3）Preparation of AteloGene® QG & siRNA/miRNA complex
While cooling on ice, gently pour 540 μL of the ②QG buffer and then 120 μL of the siRNA/miRNA solution onto 540 μL of AteloGene® QG in the ③2.0 mL microtube. Rotate and mix the solutions at 4℃ for 10 minutes. To avoid forming bubbles, the rotation speed should be approximately 12 r.p.m. (in the case of a rotator with a diameter of 20 cm).
Although the mixture may become cloudy during this procedure, it will become a clear solution as the components are thoroughly mixed by rotation.
4）Defoamation of bubbles and preparation for administration
Centrifuge the mixture at 10,000 r.p.m. for 1 minute at 4℃ to defoam bubbles. Attach the ④18G needle to the disposable syringe and slowly draw the mixture while avoiding forming bubbles. Replace the needle with the 27G needle and keep the syringe refrigerated until immediately before administration.