Revetra ace kits

Total RNA was extracted from cell lines with ISOGEN (Nippongene, Tokyo, Japan) and reverse transcription-PCR was performed using Revetra Ace Kits (Toyobo, Tokyo, Japan). Real time PCR was performed using the EvaGreen PCR Master Mix (Bio-Rad Technologies, CA, USA). The mRNA expression of target genes was normalised to GAPDH. The primer sequences were as follows: GAPDH, sense 5ʹ-TGCACCACCAACTGCT-3ʹ, antisense 5ʹ-GGCATGGACTGTGGTC-3ʹ; Ido1, sense 5ʹ-CCCACACTGAGCACGG-3ʹ, antisense 5ʹ-TTGCGGGGCAGCACCT-3ʹ; Ido2, sense 5ʹ-GCCCAGAGCTCCGTGC-3ʹ, antisense 5ʹ-TGGGAAGGCGGCATGT-3ʹ.

The total RNA was extracted from cell lines with ISOGEN (Nippon GENE, Tokyo, Japan) and the RNA concentration was determined spectrophotometrically at 260 nm. Reverse transcription-PCR was performed by using Revetra Ace Kits (TOYOBO, Japan). Primers used in semi-quantitative analysis were as follows-GAPDH: 5′-accacagtccatgccatcac (sense) and 5′-tccaccaccctgttgctgta (antisense); IDO1: 5′-cctgacttatgagaacatggacgt (sense) and 5′-atacaccagaccgtctgatagctg (antisense). Semi-quantitative analysis of RT-PCR products was performed by using NIH ImageJ 1.34 s software and normalized to GAPDH. When the same experiment was repeated three times, the three experiments showed similar results. Therefore, the results obtained from one of the three experiments were shown.