Abi prism 7900 ht sequence detection system 96

Total RNA was extracted from tissue samples using TRIzol (Invitrogen) according to the manufacturer’s protocol. Samples were treated with DNase using the RNase-free DNase Set (Qiagen) during the total RNA isolation. First strand complementary DNA (cDNA) was synthesized using the cDNA Synthesis kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. ABI prism 7900-HT sequence detection system (96-well, Applied Biosystems) was used to perform quantitative real-time PCR (RT-PCR) analysis. For RT-PCR, the following primers were used:
GAPDH: 5′- GAC​TCA​TGA​CCA​CAG​TCC​ATG​C-3′ (forward), 5′- AGA​GGC​AGG​GAT​GAT​GTT​CTG-3′ (reverse).
H1FX-AS1: 5′- GAT​GGG​GAA​GGG​ATT​CGC​TC-3′ (forward), 5′- TCT​CCT​TTG​CTG​TGT​TCC​CG-3′ (reverse).
AL441992.1: 5′- AAG​AAG​CTC​TCG​TGT​GGC​TC-3′ (forward), 5′- TGG​CTT​TGA​AGC​GAG​GAT​GA-3′ (reverse).
USP30-AS1: 5′- AGC​AAT​AGC​TGA​CGG​ACC​AC-3′ (forward), 5′- TGA​AAA​CCA​AGC​AGC​CCC​A-3′ (reverse).
AP001527.2: 5′- ATT​GGG​AAT​GAC​TCA​TCT​GTT​TG-3′ (forward), 5′- AGC​AGT​AGA​CTC​CCA​GGA​AAG-3′ (reverse).
AL031123.2: 5′- ACA​CAC​GTG​GTC​TGT​AGC​G-3′ (forward), 5′- GGG​CCT​TGC​TTT​CCC​CAT​AA-3′ (reverse).
All samples were processed in triplicate. The relative gene expression was determined using the 2^−ΔΔCT method.

To quantify PD-L1 mRNA expression, total RNA was extracted from cells with TRIzol reagent (Invitrogen), and cDNA was synthesized using TaqMan MultiScribe Reverse Transcriptase (SuperScript™ IV Reverse Transcriptase, Thermo Fish) according to the manufacturer’s instructions. Quantitative real-time PCR (RT-PCR) analysis was performed using an ABI Prism 7900-HT Sequence Detection System (96-well, Applied Biosystems). For RT-PCR, the following primers were used for amplification: PD-L1, forward primer 5′-TGGCATTTGCTGAACGCATTT-3′ and reverse primer 5′-TGCAGCCAGGTCTAATTGTTTT-3′; and GAPDH, 5′-GGAGCGAGATCCCTCCAAAAT-3′ (forward) and 5′-GGCTGTTGTCATACTTCTCATGG-3′ (reverse). The experiments were performed in triplicate. The relative expression of PD-L1 was normalized to GAPDH expression.