9700 real time pcr system

Total RNA was extracted using a commercial kit, and real-time PCR was performed with the 9700 Real-time PCR System (Applied Biosystems, Carlsbad, California, USA) and the SDS 2.1 software (Applied Biosystems). The PCR primers are shown in Supplemental Table 2. Each PCR run started with the incubation of samples at 50 °C for 2 min, followed by incubation at 95 °C for 10 min, and 40 cycles of incubation at 95 °C for 15 s and at 59 °C for 30 s. Data were normalized by quantifying the amount of amplified cDNA products by calculating the ratio of the amount of cDNA relative to the amount of GAPDH cDNA.

In total, 3 tagging single-nucleotide polymorphisms (SNPs) of IFNR (rs2069718, rs1861493, rs2069705) with a minimum allelic frequency of >10% in the Han Chinese population were selected from the HapMap database (http://hapmap.ncbi.nlm.nih.gov/). The rs2069718 was located on the 5’UTR of IFNG, and rs1861493 and rs2069705 of the IFNG gene were located in the introns. Genotyping was carried out using a TaqMan Allelic Discrimination Assay (Applied Biosystems, Foster City, CA), as previously described.^{24 (link)} Quantification of PCR was performed using a 96-well microplate with an ABI 9700 Realtime PCR system, with the following thermal cycling conditions: denaturing at 95°C for 10 minutes, followed by 40 cycles of denaturing at 92°C for 15 seconds and annealing and extension at 60°C for 1 minute, as previously described.^{24 (link)} Fluorescence was quantified using System SDS software version 1.2.3 (Applied Biosystems).

RNA was extracted using the TRIzol reagent (Life Technologies, Grand Island, NY, USA). Transcripts were amplified using a reverse transcriptase polymerase chain reaction kit (Life Technologies). Real-time PCR was performed on an Applied Biosystems 9700 real-time PCR system using gene-specific oligonucleotide primers for TaqMan probes (Applied Biosystems, Foster City, CA, USA). TaqMan probes were as follows: GAPDH (Hs99999905_m1), HIF-1α (Hs00153153_m1), and vascular endothelial growth factor (VEGF, Hs00900055_m1). For mRNA expression, gene expression was normalized t GAPDH.