Qpcr detection kit

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)].

Total RNA was isolated, and complementary DNA (cDNA) was synthesized with a Primer Script TM RT kit (Takara, Dalian, China). Targeted GAPDH (housekeeping gene), Bcl-2, Bax, Fas, caspase-3, caspase-8, and p53 primers were prepared using the Primer Premier 5.0 software (PREMIER Biosoft, Palo Alto, CA, USA) and generated by Shanghai Biotechnology Co. Ltd (Shanghai, PR China). qPCR was performed using an ABI 7500 qPCR system (Applied Biosystems, Foster City, CA, USA) and qPCR Detection Kit (Takara) as per kit instructions under the following conditions: denaturation (94 °C, 3 min), denaturation (40 cycles, 94 °C, 30 s), annealing (52–55 °C, 30 s), and elongation (72 °C, 30 s). Each experiment was repeated three times and performed in duplicate in 96-well plates. Relative gene levels were computed using the 2^−△△Ct formula, and alterations in gene levels were evaluated via the Student’s t-test (P < 0.05 was set as the significance threshold).