E gel precast agarose electrophoresis system

Total RNA in the cells was extracted using the Qiagen RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer's instruction. The RNA samples were transcribed into cDNA in a 20 μl volume, using the QuantiTect reverse transcription kit (Qiagen).The thermal cycle included the following real-time PCR conditions: 95°C for 5 min, followed by 40 cycles (denaturation for 15 sec at 95°C, annealing for 20 sec at 60°C) and extension for 20sec at 72°C). The primers sequences and the size of the PCR product for the target and reference gene are listed in supplementary results table S1. The expression levels of mRNA of different markers were detected by real-time PCR with ß-actin as internal reference, using Mesa Blue qPCR Master Mix Plus for SYBR assay (Eurogentec) on the Master cycler Realplex2 (Eppendorf).The PCR products of cell lines and tissue samples, after real-time PCR, were electrophoresed by E-Gel Precast Agarose Electrophoresis System (Thermo Fisher Scientific Inc., Waltham, USA).

Total RNA (5 μg) was reverse transcribed using the miRCURY LNA RT Kit (QIAGEN). Poly(A) tails were added to mature miRNA templates, and cDNA synthesized using a 5′ universal tag and 3′ degenerate anchor. The resultant cDNA was used for LNA-based RNA expression detection using a miRCURY LNA SYBR Green PCR Kit (QIAGEN) on an ABI StepOnePlus system (Applied Biosystems). U3-derived miRNA relative expression was calculated by ΔΔCT-method Ct with U6 as reference using custom made primers synthesized by QIAGEN. The amplified products were also visualized by electrophoresis using the E-Gel Precast Agarose Electrophoresis System (Thermo Fischer Scientific).