1. Preparation of miRNA1.1 The miRNA product is a transparent or translucent dry film attached to the tube wall. Centrifuge the tube briefly before use to ensure that the miRNA is at the bottom of the tube. Reconstitute the miRNA with nuclease-free water to obtain a 20 μM stock solution. For 5 nmol miRNA: add 250 μL nuclease-free water and vortex several times to dissolve. For 20 nmol miRNA: add 1000 μL nuclease-free water and vortex several times to dissolve. 1.2 (Optional) Aliquot the miRNA into one or more tubes to reduce the number of freeze-thaw cycles (<5). 1.3 Store the prepared stock solution at -20℃ or -80℃. 2. Prepare cells2.1 Seed the cells in advance and transfect the cells when the appropriate cell density is reached. Cell viability before transfection affects the transfection results. 3. Transfection3.1 Prepare transfection mixture A and B Note: For miRNA mimics, we recommend a working concentration of 50nM. The optimal working concentration of miRNA products varies with different cell types and research purposes, depending on the miRNA, cell line, and selected analysis method. In order to determine the concentration for the best results, optimization experiments should be performed using different concentrations. The optimized concentration range is 10~200nM. Each well of a 6-well plate: A: 245 μL serum-free medium + 5 μL miRNA; B: 240 μL serum-free medium + 10 μL HY-K2017 siRNA/miRNA Transfection Reagent Each well of a 12-well plate: A: 97.5 μL serum-free medium + 2.5 μL miRNA; B: 95 μL serum-free medium + 5 μL HY-K2017 siRNA/miRNA Transfection Reagent Each well of a 24-well plate: A: 48.75 μL serum-free medium + 1.25 μL miRNA; B: 47.5 μL serum-free medium + 2.5 μL HY-K2017 siRNA/miRNA Transfection Reagent Each well of a 96-well plate: A: 24.75 μL serum-free medium + 0.25 μL miRNA; B: 24.5 μL serum-free medium + 0.5 μL HY-K2017 siRNA/miRNA Transfection Reagent If you use a transfection reagent from another brand, you need to adjust the amount of transfection reagent added according to the specific situation. 3.2 Gently mix the diluted A and B. Incubate at room temperature for 15-20 minutes. 3.3 Remove the culture medium from the cells and wash with PBS. 3.4 Add the miRNA-transfection reagent mixture to the cells. To each well of a 6-well plate: add 1500 μL of fresh culture medium without serum, then add 500 μL of transfection mixture (A+B), and mix well. To each well of a 12-well plate: add 800 μL of fresh culture medium without serum, then add 200 μL of transfection mixture (A+B), and mix well. To each well of a 24-well plate: add 400 μL of fresh culture medium without serum, then add 100 μL of transfection mixture (A+B), and mix well. To each well of the 96-well plate: add 50 μL of fresh medium without serum, then add 50 μL of transfection mixture (A+B) and mix well. 3.5 Incubate the cells at 37°C for 1-3 days. Then analyze the transfected cells. The medium without double antibody and serum can be replaced with normal medium containing serum and double antibody about 6 hours after transfection. Note: Do not add antibiotics to the medium during transfection, otherwise it will reduce the efficiency of cell transfection and even cause cell death.

13928.39

White to off-white solid powder

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)