Superscript m mlv reverse transcriptase

RT-qPCR was performed as described before [24 (link)]. Briefly, total RNA or miRNA from cultured ESCs and tissues was extracted by TRIpure (RP1001, BioTeke, China). Then, total miRNA was reversed transcribed using the miRNA First Strand cDNA Synthesis kit (#B532451, Sangon, Shanghai). Total RNA was converted into first-strand cDNA using SuperScript M-MLV reverse transcriptase (PR6502, BioTeke) with Exicycler^TM 96 Real-Time Quantitative Thermal Block (BIONEER, Korean). RT-qPCR was used to examine miR-106a-5p and FOXC1 expression using an SYBR Green Master Mix (Solarbio, Beijing). β-actin was served as an internal control. The relative expression of miR-106a-5p and FOXC1 was calculated in a 2^−∆∆CT manner. The primer sequences were exhibited as followed: hsa-miR-106a-5p, forward: 5ʹ- AAAAGTGCTTACAGTGCAGGTAG −3ʹ; FOXC1, forward: 5ʹ- ACAGCATCCGCCACAACCTC −3ʹ, reverse: 5ʹ- TGTCCTTCACCGCGTCCTTC −3ʹ; β-actin, forward: 5′- CACTGTGCCCATCTACGAGG-3′, reverse: 5′- TAATGTCACGCACGATTTCC-3′.