qRT-PCR

Total RNA was isolated from tissue specimens and cultured cells by using a modified Trizol co-purification technique.¹⁶ The levels of circ_0031242 and POU3F2 mRNA were quantified using PrimeScript^TM One-Step TB Green^® RT-PCR Kit (TaKaRa, Dalian, China) in a 20 μL of reaction containing 100 ng of total RNA with primers specific for circ_0031242: 5ʹ-TACCATTGGCCTCTAGCCCT-3ʹ (forward) and 5ʹ-CAAGGGGAATCACAGCCCAT-3ʹ (reverse), or POU3F2: 5ʹ-TGGGATTTACCCAAGCGGAC-3ʹ (forward) and 5ʹ-CGCCTGCAAAGGTCAAAACA-3ʹ (reverse). Quantification of miR-924 was carried out using miRCURY LNA^TM Universal RT miRNA PCR (Exiqon, Denmark) with primer for miR-924: 5ʹ-TCTTCTCTGTTTTGGCCA-3ʹ (forward) and 5ʹ-GAACATGTCTGCGTATCTC-3ʹ (reverse). Each reaction was individually run on a 480 II Light Cycler (Roche, Basel, Switzerland) in triplicate. Results were calculated using the 2^−ΔΔCt method and normalized based on the expression of β-actin: 5ʹ-CTCGCCTTTGCCGATCC-3ʹ (forward) and 5ʹ-GGGGTACTTCAGGGTGAGGA-3ʹ (reverse), or U6: 5ʹ-CTCGCTTCGGCAGCACATATACT-3ʹ (forward) and 5ʹ-ACGCTTCACGAATTTGCGTGTC-3ʹ (reverse). Reactions were incubated for 10 min at 95°C, and then for 40 cycles at 94°C for 15 s, and 60°C for 60 s.