Taqman probes for specific genes

Total RNA was isolated from tumor tissue using Purelink RNA Minikits (Ambion, Life Technologies) according to the manufacturer’s instructions. The quality and quantity of RNA samples were measured using a spectrophotometer (Amersham Biosciences, Little Chalfont, UK). An A260/A280 ratio of 1.8–2.0 for RNA was considered suitable for real time qPCR. cDNA was prepared using the Reverse Transcriptase Mix (Qiagen Ltd.). Quantitative PCR was carried out in accordance with the RT² Profiler PCR array instructions. Cancer Inflammation and Immunity Crosstalk PCR Arrays (Qiagen) were used to analyze the expression of 84 immune-related genes, a subset of which (CTLA-4, FOXP3, granzyme B, and IDO1) were selected for further study by real time quantitative PCR using the Taqman probes for specific genes (Applied Biosystems) according to manufacturer’s instructions. The relative mRNA values were calculated by double delta method and normalized to the level of housekeeping gene GusB.

RNA was isolated using Trizol reagent (Invitrogen), following manufacturer’s instructions. Briefly, the cells were lysed with Trizol, followed by phenol-chloroform phase separation. RNA was precipitated with isopropanol, washed with 75% ethanol and dried RNA pellet was resuspended in DEPC water. RNA from mouse tissue was isolated using RNeasy mini kit (Invitrogen), according to the instruction manual. cDNA was synthesized using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems). Gene expression levels were assayed by RT-qPCR using 7900 HT FAST Real-Time PCR system, TaqMan Fast Universal Master Mix and TaqMan probes for specific genes (Applied Biosystems): ITGAV Hs00233808_m1, ITGB3 Hs00173978_m1, MYC Hs00153408_m1, SERPINE (PAI-1) Hs01126607_g1, GAPDH Hs99999905_m1. All assays were performed in triplicate and analysis was performed using RQ Manager software (Applied Biosystems) and the 2^−ΔΔCt method to obtain relative quantitation (RQ) values, with GAPDH used as endogenous control.