Sybr green qpcr supermix udg reagents

Total RNA was extracted using TRIzol reagent (Invitrogen, USA). Nuclear and cytoplasmic RNA was extracted and purified by the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Invitrogen, USA) and following the manufacturer’s protocols. Then, reverse transcription of RNA into cDNA was carried out by M-MLV reverse transcriptase (Promega, USA). Real-time PCR assays were carried out by SYBR Green qPCR SuperMix-UDG reagents (Invitrogen, USA) with a CFX96 Touch sequence detection (Bio-Rad, USA). GAPDH was used as the endogenous control for genes mentioned in this article. First, results of the QPCR were normalized through the internal reference gene (GAPDH) and then calculated the relative quantification by the formula of 2^一△△CT. All the final results came from three replicates. All primer sequences were provided in Supplemental Table 1.

TRIzol reagent (Invitrogen) was used to isolate total RNA from monocytes and macrophages as described above. The concentration and quality of the total RNA were determined using NanoDrop 2000. First-strand complementary DNA was synthesized using random primers and the M-MLV reverse transcriptase (Promega). PCR was performed in triplicate using SYBR Green qPCR SuperMix-UDG reagents (Invitrogen) on a CFX96 Touch sequence detection system (Bio-Rad). GAPDH was used as an endogenous control for all the genes. The primer sequences are listed in Supplementary information, Table S10.

The PCR reactions were done on a Bio-Rad CFX96 sequence detection system (Bio-Rad Laboratories Inc., Hercules, CA, USA) by pre-incubating at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 30 s in a 20 μl reaction volume containing 2 μl reverse transcription product, 9 μl SYBR Green qPCR SuperMix-UDG reagents (Invitrogen), 4 μl PCR forward and reverse primer mix, and 5 μl H₂O₂. Primers for miR-24 and U6 amplification were purchased from RiboBio. Primers for FSCN1 amplification were listed as follows: 5′-CTACAACATCAAAGACTCCACAG-3 (forward) and 5′-ATGGCCACCTTGTTATAGTC-3′ (reverse); and primers for GAPDH amplification were: 5′-CTCCTCCTGTTCGACAGTCAGC-3′ (forward) and 5′-CCCAATACGACCAAATCCGTT-3′ (reverse). All of the reactions were performed in triplicate. The U6 or GAPDH were used as normalized controls, and the relative levels were calculated using the 2^-ΔΔCT equation.