IDO mRNA Expression in Breast Cancer

The mRNA expression of IDO gene in breast cancer cell lines was analyzed using RT-PCR. Trizol Reagent (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA, and MMLV reverse transcriptase (Promega, Madison, WI, USA) was used to reverse transcribe to cDNA. Expression levels of target genes were quantified using the SYBR Premix Ex Taq system (Takara Bio, Tokyo, Japan) following the manufacturer’s instructions. The primers applied in this assay were: IDO (188 bp), sense 5′-CATCTGCAAATCGTGACTAAG-3′; antisense 5′-CAGTCGACACATTAACCTTCCTTC-3′. β-actin (186 bp) was used as an internal control; sense 5′-TGGCACCCAGCACAATGAA-3′; antisense 5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′. The specificity of the primers was verified and described in our prior papers (17 (link), 18 (link)). The thermal cycling program was listed below: initial denaturalization at 94°C for 5 min, then 94°C for 30 s, 58°C for 30 s, and 72°C for 45 s for 35 cycles; after the last cycle, 72°C for 10 min. All tests were repeated at least three times.