miRNA-9 Modulation in Macrophages

The miRNA-9 mimics and inhibitors were transfected into macrophages RAW264.7. An appropriate amount of Trizol reagent was added to the treated cells to lyse the cells. After the chloroform reagent was added for 10 minutes, the solution was centrifuged at low temperature and high speed for 15 minutes to collect the supernatant. After adding an equal volume of isopropanol and mixing, the supernatant was allowed to stand at room temperature for 5 minutes, and then centrifuged at low temperature and high speed for 10 minutes to take the precipitate. The precipitate was washed with a pre-cooled 75% ethanol solution and dried naturally, added an appropriate amount of RNase-free ultrapure water to dissolve, and stored in a refrigerator at −80°C. Reverse transcription of complementary deoxyribonucleic acid (cDNA) was performed according to the instructions of the cDNA reverse transcription kit (Takara, Japan). The cDNA was undertaken as a template, and the target gene was quantified according to the instructions of the qPCR detection kit (Takara, Japan). With glyceraldehyde-3phosphate dehydrogenase (GAPDH) gene as an internal reference, the relative expression level of the target gene was calculated according to 2^−ΔΔCT [8 (link)].