1. miRNA Preparation 1.1 The miRNA product is a transparent or translucent dry film adhering to the tube wall. Before use, briefly centrifuge the tube to ensure the miRNA is at the bottom. Reconstitute the miRNA using nuclease-free water to obtain a 20 μM stock solution. For 5 nmol miRNA: add 250 μL of nuclease-free water and vortex several times to dissolve. For 20 nmol miRNA: add 1000 μL of 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. Cell Preparation 2.1 Pre-seed cells and perform transfection when the appropriate cell density is reached. Cell viability before transfection affects the transfection results. 3. Transfection 3.1 Preparation of Transfection Mixtures A and B Note: For miRNA inhibitors, we recommend a working concentration of 100 nM. The optimal working concentration of miRNA products varies depending on the cell type and research objective, as well as the miRNA, cell line, and chosen analytical method. To determine the concentration for optimal results, experiments should be optimized using different concentrations. The optimized concentration range is 20–500 nM. For each well of a 6-well plate: A: 240 μL serum-free medium + 10 μL miRNA; B: 230 μL serum-free medium + 20 μL siRNA/miRNA Transfection Reagent. For each well of a 12-well plate: A: 95 μL serum-free medium + 5 μL miRNA; B: 90 μL serum-free medium + 10 μL siRNA/miRNA Transfection Reagent. For each well of a 24-well plate: A: 47.5 μL serum-free medium + 2.5 μL miRNA; B: 45 μL serum-free medium + 5 μL siRNA/miRNA Transfection Reagent. For each well of a 96-well plate: A: 24.5 μL serum-free medium + 0.5 μL miRNA; B: 24 μL serum-free medium + 1 μL siRNA/miRNA Transfection Reagent. If using other brands of transfection reagents, the amount added needs to be adjusted 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. For each well of a 6-well plate: add 1500 μL of serum-free fresh culture medium, then add 500 μL of the transfection mixture (A+B), and mix well. For each well of a 12-well plate: add 800 μL of serum-free fresh culture medium, then add 200 μL of the transfection mixture (A+B), and mix well. For each well of a 24-well plate: add 400 μL of serum-free fresh culture medium, then add 100 μL of the transfection mixture (A+B), and mix well. For each well of a 96-well plate: add 50 μL of serum-free fresh medium, then add 50 μL of transfection mixture (A+B) and mix well. Incubate cells at 37°C for 1–3 days. Then analyze the transfected cells. Approximately 6 hours after transfection, the serum-free and antibiotic-free medium can be replaced with normal medium containing serum and antibiotics. Note: Do not add antibiotics to the transfection medium, as this will reduce transfection efficiency and may even lead to cell death.

Blue to dark blue solid powder

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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.)