Total RNA was extracted from LSCC specimens and cell lines of AMC-HN-8 and TU177 with Eastep®Super Total RNA Extraction Kit (Promega, USA) following the experimental instructions. RNA reverse transcription into cDNA was performed with Transcriptor First Strand cDNA Synthesis Kit (Roche, Germany). A GoTaq®qPCR Master Mix (Promega, USA) was used for the quantitative real-time PCR (qRT-PCR). Relative expression was normalized using the 2−∆∆Ct method and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was assigned into the endogenous control. Primer sequences were as follows:
PRAME, forward: 5′-CAGGACTTCTGGACTGTATGGT-3′;
reverse: 5′-CTACGAGCACCTCTACTGGAA-3′.
GAPDH, forward: 5′-CTCACCGGATGCACCAATGTT-3′;
reverse: 5′-CGCGTTTCACAATGTTCAT-3′.
All the samples were run in triplicate.
PRAME, forward: 5′-CAGGACTTCTGGACTGTATGGT-3′;
reverse: 5′-CTACGAGCACCTCTACTGGAA-3′.
GAPDH, forward: 5′-CTCACCGGATGCACCAATGTT-3′;
reverse: 5′-CGCGTTTCACAATGTTCAT-3′.
All the samples were run in triplicate.
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