MicroRNA Inhibitor Negative Control

1. Preparation of miRNA 1.1 A transparent or translucent dry film that is affixed to the tube wall is the form of the miRNA product. Before using, give the tube a quick centrifugation to make sure the miRNA is at the bottom. Reconstitute the miRNA in nuclease-free water to provide a stock solution with a concentration of 20 μM. To dissolve 5 nmol of miRNA, add 250 μL of nuclease-free water and vortex many times. To dissolve 20 nmol of miRNA, add 1000 μL of nuclease-free water and vortex many times. 1.2 (Optional) To cut down on the amount of freezing and thawing times (<5), dispense the miRNA into one or more tubes. 1.3 Keep the ready mother solution between -20°C and -80°C. 2. Cell preparation 2.1 Sow seeds first, and then, once the desired cell density is attained, carry out cell transfection. Results of transfection are influenced by cell viability prior to transfection. 3. Transfection 3.1 Making transfection mixes A and B Observation: We advise using 100 nM of miRNA inhibitors as your working concentration. The ideal concentrations of miRNA products for various cell types and research objectives vary based on the selected miRNA, cell line, and analytic technique. Optimization tests should be carried out utilizing various concentrations in order to ascertain the concentration that yields the optimum outcomes. The range of concentrations that is optimal is 20~500nM. In each well of the six-well plate, there is: B: 121 μL serum-free media + 4 μL PolyFast transfection reagent; A: 120 μL serum-free medium + 5 μL miRNA. The contents of each well in the 24-well plate are as follows: B: 24 μL serum-free medium + 1 μL PolyFast transfection reagent; A: 23.75 μL serum-free medium + 1.25 μL miRNA. The contents of each well in the 96-well plate are as follows: B: 4.8 μL serum-free medium + 0.2 μL PolyFast transfection reagent; A: 4.75 μL serum-free medium + 0.25 μL miRNA. If you use transfection reagents from a different brand, you must modify the amount added based on the particular circumstance. 3.2 Combine the weaker versions of A and B. For fifteen minutes, incubate at room temperature. 3.3 Take out the culture medium and give the cells a PBS wash. 3.4 Give the cells the mixture of miRNA-transfection reagents. Add 250 μL of the transfection mixture (A+B) and mix thoroughly after adding 1750 μL of new culture media without double antibody to each well of the 6-well plate. with each well of the 24-well plate, add 50 μL of the transfection mixture (A+B) and well mix with 450 μL of new culture media without double antibodies. Put 10 μL of the transfection mixture (A+B) and mix thoroughly after adding 90 μL of new culture media without double antibodies to each well of the 96-well plate. 3.5 Let cells sit at 37°C for one to three days. After that, transfected cells were examined.

6953.56

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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.

---

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).

View More

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)

---

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).

View More

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

---

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.

---

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