Abi 7900 ht real time pcr system

RNAiso Plus was used to isolate total RNA from 293T and SHY5Y cells and from mouse hippocampi. The RNA was measured spectrophotometrically based on the absorbance at 260 nm. One microgram of RNA was used as a template for quantitative reverse transcriptase- (RT-) PCR amplification using One Step SYBR® PrimeScrip™ RT-PCR Kit (TaKaRa). Real-time PCR reactions were performed using an ABI 7900HT Real-Time PCR System with SYBR® Premix Ex Taq™ II (TaKaRa). The relative abundance of transcripts was calculated based on normalization to the GAPDH gene. The primers are shown in Table 2.

Total-RNA was extracted using the RNeasy kit (Beyotime, Shanghai, China, R0027) according to the manufacturer’s directions. First-strand cDNA was synthesized from an RNA template (1 μg) using SuperScript II reverse transcriptase (TaKaRa, Japan, RR047). Real-time PCR was then performed using the SYBR Green Mix (TaKaRa, Japan, RR820) on an ABI 7900 HT Real-Time PCR system in duplicate. The primer sequences were as follows: IDO1, 5′-GAAAGCTCTTCTGAGTTGGCCT-3′ (forward) and 5′-GATGAAGGTGTTTTCTGTGCCC-3′ (reverse); CD8A, 5′-AGGCCCTCTCCCATGTCTAA-3′ (forward) and 5′- CGGGGGTGCTAAGGAATGTT-3′ (reverse); PD-1, 5′- ACTGCTACTGAAGGCGACAC-3′ (forward) and 5′- AGCCCAAGTGAATGACCAGG-3′ (reverse); programmed death ligand 2 (PD-L2), 5′-ATTGCATGGGCTTTGTGCTC-3′ (forward) and 5′-ACCACGGGCAAGCTTTTATTC-3′ (reverse); TIM3, 5′-TGTGCTCAAGGGGAACTGAC-3′ (forward) and 5′-ACTCTGCCTTCGTATGTCC-3′ (reverse); CD276, 5′-ACCTTGCTTCCGACTTACCC-3′ (forward) and 5′-GGGCCATGCTTTCTCCATGT-3′ (reverse); CD200, 5′-ACCCCAGCTTCTTTTTCTGTGA-3′ (forward) and 5′-TGTCTTCAGAACAAAGCGATTGTA-3′ (reverse); CD160, 5′-AGCCCGTGAACTTTCGTGTA-3′ (forward) and 5′-AGCTCAGTGGCTTCACAAAT-3′ (reverse); GADPH, 5′-TGGAGAAACCTGCCAAGTATGA-3′ (forward) and 5′-GGTCCTCAGTGTAGCCCAAG-3′ (reverse). All of the data were normalized to GADPH mRNA.