1. miRNA Resuspension 1.1 Briefly centrifuge the tube to ensure the dried miRNA precipitates at the bottom. 1.2 Resuspend the miRNA in nuclease-free water to prepare a 20 μM stock solution. 5 nmol miRNA: Add 250 μL nuclease-free water. 20 nmol miRNA: Add 1000 μL nuclease-free water. 1.3 Aliquot the miRNA into one or more tubes to minimize freeze-thaw cycles (<5 times). 1.4 Store at -20°C or below in a non-frost-free freezer until use. 2. Cell Preparation 2.1 Seed cells in advance for transfection. Cell viability and overall health before transfection significantly affect the transfection results. 3. Transfection 3.1 Prepare transfection mixtures A and B. 6-well plate per well: A: 240 μL serum-free medium + 10 μL miRNA; B: 230 μL serum-free medium + 20 μL siRNA/miRNA transfection reagent. 12-well plate per well: A: 95 μL serum-free medium + 5 μL miRNA; B: 90 μL serum-free medium + 10 μL siRNA/miRNA transfection reagent. 24-well plate per well: A: 47.5 μL serum-free medium + 2.5 μL miRNA; B: 45 μL serum-free medium + 5 μL siRNA/miRNA transfection reagent. 96-well plate per well: A: 24.5 μL serum-free medium + 0.5 μL miRNA; B: 24 μL serum-free medium + 1 μL siRNA/miRNA transfection reagent. Note: The recommended working concentration of the miRNA inhibitor is 100 nM. The function of miRNAs may vary depending on the miRNA species, cell line, and chosen analytical method. To determine the optimal concentration, experiments should be optimized using different concentrations of mimics/inhibitors. An optimization range of 20 to 500 nM is recommended for miRNA concentrations. If using other transfection reagents, the amount of transfection reagent needs to be adjusted accordingly. 3.2 Gently mix A and B. Incubate at room temperature for 15 minutes. 3.3 Remove the cell culture medium and wash with PBS. 3.4 Add the transfection mixture (A+B) to the cells. For each well in a 6-well plate: Add 1500 μL of serum-free medium, then add 500 μL of transfection mixture (A+B), and mix thoroughly. For each well in a 12-well plate: Add 800 μL of serum-free medium, then add 200 μL of transfection mixture (A+B), and mix thoroughly. For each well in a 24-well plate: Add 400 μL of serum-free medium, then add 100 μL of transfection mixture (A+B), and mix thoroughly. For each well in a 96-well plate: Add 50 μL of serum-free medium, then add 50 μL of transfection mixture (A+B), and mix thoroughly. 3.5 Incubate cells at 37°C for 1–3 days. Then analyze the transfected cells. If necessary, replace with fresh serum-containing medium after 6 hours. Note: Antibiotics increase toxicity and should therefore be avoided during transfection. Medium containing polyanions such as heparin, heparin sulfate, or dextran sulfate will inhibit transfection.

[1]. Xia W, Huang J, Sun C, Shen F, Yang K. MicroRNA‑1224 inhibits cell proliferation by downregulating CBX3 expression in chordoma. Oncol Lett. 2024;27(6):262.

Light yellow to yellow solid

[FAM-MicroRNA Mimic Negative Control]

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: 请将本产品存放在密封且受保护的环境中（例如氮气下），避免暴露在潮湿和光照下。

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.)