Modelling Intratumoral Treatment with miRNA Inhibitors: A Method to Analyze the Effect of miRNA Inhibitors on Tumor Growth in Murine Model

After establishing a Cal62 human thyroid cancer cell line in complete medium, suspend a 1 times 10 to the sixth cell aliquot in 50 microliters of PBS at 4 degrees Celsius and mix the cells with an equal volume of basement membrane matrix.

Load the cells into a 1-milliliter syringe equipped with a 27G 1/2" needle and subcutaneously inject 100 microliters of the sample into the left flank of a 6-week-old immunodeficient BALB/c nude mouse. Two weeks after the injection, add the antagomiR or control treatment buffer solution to 160 microliters of room temperature in vivo delivery reagent in a 1.5-milliliter tube and immediately vortex the solution for 10 seconds to ensure complexation of the mixture.

Incubate the treatment solution for 30 minutes at 50 degrees Celsius, followed by a brief centrifugation in a microcentrifuge. Then, dilute the sedimented treatment complex six-fold with fresh PBS and thorough mixing, and inject the entire 200-microliter volume of the treatment directly into the tumor.

Inject 50 microliters of a 40 milligrams per milliliter D-luciferin substrate solution subcutaneously two times a week into each experimental animal, making sure to confirm a lack of response to toe pinch after isoflurane anesthesia. Place the animal in the chamber of an in vivo bioluminescence imaging system and image the bioluminescent signal with the in vivo imaging software according to standard protocols.