Sybr premix ex taq 1

Total RNA was isolated from treated-microglial cells by using Trizol reagent (Invitrogen Life Technologies, California, USA). The quality and quantity of total RNA were measured by NanoDrop 2000 spectrophotometer (NanoDrop Technologies, Thermo Scientific, USA). According to the manufacturer's instructions, 1 μg total RNA was reverse-transcribed and synthesized the complementary DNA by using PrimeScriptTM RT Master Mix (TaKaRa, Japan). Real-time qPCR was carried out using SYBR^® Premix Ex Taq I (TaKaRa, Japan) following the instructions of reagent kit on a Quant Studio 5 Real-Time PCR System (Applied Biosystems, USA). The primer sequences of target genes were shown in Table 1. GAPDH was regarded as the internal standard to normalize other target genes, and the 2(–ΔΔct) method was used to analyzed the relative expression of the target genes. Each sample was run in triplicate, and each experiment was repeated at least thrice.

Transfection was conducted with Lipofectamine 2000 (Invitrogen) following the protocol of the manufacturer. Generally, 5 × 10⁵ cells were seeded into 6-well plates one day before transfection. When cells reached the 80% confluence, the plasmid DNA and Lipofectamine 2000 were diluted with the Opti-MEM I Reduced Serum Medium, respectively, and then mixed together. After incubating for 20 minutes at room temperature, the mixture was added into the plate drop by drop. After 24–48 h, the cells were used for further experiments.
TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract RNA from 5 × 10⁵ cells following the protocol of the manufacturer. Total RNA was finally suspended in 20 µL of RNase-free water. The purity and quality of the isolated RNA was evaluated by NanoDrop with A₂₆₀/A₂₈₀ ratio of 1.9–2.0. A total of 500 ng RNA was used to synthesize cDNAs using the PrimeScript reverse transcriptase reagent kit (TaKaRa, Shiga, Japan), according to the protocol of the manufacturer. qRT-PCR was performed with the Roche Light Cycler 480 II using the SYBR Premix Ex Taq I (TaKaRa, Japan). Each reaction contained 2 μL of cDNA in a total volume of 20 μL. Relative RNA abundances were calculated by the standard 2^-ΔΔCt method after normalization to GAPDH. The specific primers used in the article are provided in Supplementary Table 1.

The following were used in the study: Dulbecco’s modified Eagle’s medium (DMEM), lipofectamine 2000, pmirGLO plasmid, Fetal bovine serum (FBS), pcDNA3.1 plasmid and Trizol reagent (Invitrogen, Carlsbad, US); matrigel (BD, New Jersey, US); SurePrep™ Nuclear or Cytoplasmic RNA Purification Kit, PrimeScrip™ RT Master Mix, SYBR Premix Ex Taq I and PrimeScript miRNA cDNA Synthesis Kit (TaKaRa, Shiga, Japan); protease inhibitor cocktail (Sigma, St. Louis, US); Dual-Luciferase Reporter Assay System (Promega, Madison, US); CCK-8 test kit (Dojindo Corp, Kyushu, Japan); antibodies against WIF1(#5652), Ki-67(#9449) and β-Actin (#4967) (Cell Signaling Technology, Danvers, US).