Total rna extraction reagent

Total RNA was obtained using Total RNA Extraction Reagent (Promega). Thereafter, mRNA expression levels were analyzed by real-time PCR using the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems) (Liu et al., 2014 (link)). Expression values were normalized using β-actin (ACTB gene). Supplementary Table S1 shows the basic information on primers, gene name, gene ID and NCBI reference sequence.

Total RNA was extracted from tissues or cells utilizing total RNA extraction reagent (Promega, Madison, Wisconsin, USA), which was then reverse transcribed into cDNA via reverse transcription reaction kit Takara, Japan). qRT-PCR was presented via ABI7500 quantitative PCR system (Applied Biosystems, Warring ton, UK) and SYBR Green PCR Kit (Takara). Relative expression levels were determined with the 2^-ΔΔCt method 8 (link). Primers were designed and synthesized by Sangon Biotech (Shanghai, China). Primer sequences were as follows: ZFPM2-AS1: 5'-CAATGGGACTAAGCCAGGCA-3' (forward), 5'-GGGCTCCACCAACAACCATA-3' (reverse); miR-515-5p: 5'-TTCTCCAAAAGAAAGCACTTTCTG-3'; TUSC3: 5'-GGCTCAGTTTGTGGCAGAATC-3' (forward), 5'-CATCGCCTTTCGAAGTTGCT-3' (reverse); GAPDH: 5'-GTCAACGGATTTGGTCTGTATT-3' (forward), 5'-AGTCTTCTGGGTGGCAGTGAT-3' (reverse).