Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR)

According to the user’s guidebook of RNAiso Plus (TaKaRa, Dalian, China), total RNA from tissues and cells were isolated. Subsequently, the extracted RNA was reverse-transcribed to the first-strand complementary DNA (cDNA) using the PrimeScript RT Reagent Kit (TaKaRa) and microRNA First-Strand cDNA Synthesis Kit (Fulen Gene, Guangzhou, China). On an ABI PRISM7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA), RT-qPCR was conducted in line with SYBR Green PCR Kit (Takara). The 2^–ΔΔCt method was used to calculate the obtained data, with glyceraldehyde 3-phosphate dehydrogenase (GAPDH for circSPATA6 and TRAF6) and U6 (for miR-182) as the internal reference. The primer sequences are presented in Table 1.

The Sequences of Primers for RT‑PCR Used in This Study