Thermoscript first strand cdna synthesis kit

Total RNA was isolated from the cells using an EASYspin Plus Tissue/Cell RNA extraction kit (Aidlab Biotechnologies Co., Ltd., Beijing, China). RNA was reverse transcribed to cDNA using the ThermoScript First Strand cDNA Synthesis kit (Aidlab Biotechnologies Co., Ltd.) according to the manufacturer's instructions. Semi-quantitative reverse transcription-PCR was performed using KOD-Plus-Neo DNA polymerase (Toyobo Biotech Co., Ltd., Shanghai, China) to investigate the complete knockdown of NSD3. Each PCR regime consisted of initial denaturation at 94°C for 2 min followed by 22 cycles (for ACTB), 32 cycles (for NSD3) at 98°C for 10 sec, 55°C for 30 sec and 68°C for 45 sec. The primer sequences were: 5′-TTGGCTTGACTCAGGATTTA-3′ and reverse 5′-ATGCTATCACCTCCCCTGTG-3′ for β-actin (ACTB); and 5′-CCATGCAGAGAAAGCATTGA-3′ and 5′-TCTTCCTCTTCCGCACTTGT-3′ for NSD3. qRT-PCR was conducted using SYBR Premix Ex Taq™ (Takara, Dalian, China) at 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 34 sec in the ABI StepOnePlus Real-time PCR system. Relative gene expression was quantified relative to the GAPDH level using the comparative cycle threshold (Ct) method. Each sample was analyzed in duplicate.