Quantitative Real-Time PCR Protocol for SCLC

Quantitative real-time PCR (RT-qPCR) were performed according to methods published previously [23 (link), 24 (link)]. Total RNA was extracted from the PPFE samples by using RNeasy FFPE Kit (cat. no. 73504, QIAGEN, Germany). Total RNA of 1 μg was reversely transcribed in a 20 μl reaction using RevertAid First Strand cDNA Synthesis Kit (cat. no. #K1622, Thermo Scientific, USA) according to the manufacturer’s protocol. The reaction products were then diluted with 40 μl RNase-free water. The real-time PCR reaction was composed of 2 μl cDNA, 10 μl of PowerUp^™ SYBR^™ Green Master Mix (cat. no. A25741, Thermo Scientific, USA) and 0.5 μl of forward and reverse primers (0.5 μM). RT-qPCR was conducted in an ABI Prism 7500 analyzer (Applied Biosystems, USA) for 40 cycles (95°C for 15 sec, 58°C for 15 sec, 72°C for 30 sec) after an initial 120s denaturation at 95°C. HPRT1 was endogenous reference gene. All reactions were run in triplicate. The relative RNA levels of SCLC samples were calculated by using the 2^−ΔΔCt method. All primers of the hub genes and HPRT1 were synthesized by Sangon Biotech (Shanghai, China), and the information of their sequences were listed in Table 2.