Pe prism 7000

Single-strand cDNA was synthesized with an RT-PCR kit (Qiagen) according to the manufacturer’s instructions. Reverse transcription was performed using the RNA polymerase chain reaction (PCR) core kit (Applied Biosystems). Quantitative real-time PCR used SYBR green technology (Applied biosystems) on cDNA generated from the reverse transcription of purified RNA. After preamplification (95°C for 2 min), the PCRs were amplified for 40 cycles (95°C for 15s and 60°C for 60s) in a sequence detection system (PE Prism 7000; Perkin-Elmer Applied Biosystems). The exponential phase, linear phase and plateau phase of PCR amplification were carefully monitored to ensure a measurement of real time transcription (33). mRNA expression was normalized against GAPDH expression using the standard curve method.
PD-1-FW, 5-CCGCCTTCTGTAATGGTTTGA-3;
PD-1-RV, 5-GGGCAGCTGTATGATCTGGAA-3;
Tbet-FW, 5-GATCGTCCTGCAGTCTCTCC-3;
Tbet-RW, 5-AACTGTGTTCCCGAGGT GTC-3;
GAPDH-FW, 5-CAACAGCAACTCCCACTCTTC-3;
GAPDH- RW, 5- GGTCCAGGGTT TCTTACTCCTT-3

Single-strand cDNA was synthesized with an RT-PCR kit (Qiagen) according to the manufacturer’s instructions. Reverse transcription was performed using the RNA polymerase chain reaction (PCR) core kit (Applied Biosystems). Quantitative real-time PCR used SYBR green technology (Roche) on cDNA generated from the reverse transcription of purified RNA. After preamplification (95 °C for 2 min), the PCRs were amplified for 40 cycles (95 °C for 15 s and 60 °C for 60 s) in a sequence detection system (PE Prism 7000; Perkin-Elmer Applied Biosystems). The exponential phase, linear phase and plateau phase of PCR amplification were carefully monitored to ensure a measurement of real-time transcription [30 (link)]. mRNA expression was normalized against GAPDH expression using the standard curve method.
PD-1-FW, 5-CCGCCTTCTGTAATGGTTTGA-3;
PD-1-RV, 5-GGGCAGCTGTATGATCTGGAA-3;
LAG-3-FW, 5-CTACAACTCACCGCGTCATTT-3;
LAG-3-RW; 5-GCTCCAGACCCAGAACCTT-3;
CXCR3-FW, 5-GCCTTCCTGCTGGCTTGTAT-3;
CXCR3-RW; 5-TAGCTGCAGTACACGCAGAG-3;
TIGIT-FW, 5-GGCATGTCGCTTCAGTCTTC-3;
TIGIT-RW; 5-CTCCCCTTGTAAATCCCACC-3;
GAPDH-FW, 5-CAACAGCAACTCCCACTCTTC-3;
GAPDH-RW, 5-GGTCCAGGGTT TCTTACTCCTT-3