Quantitative RT-PCR Analysis of miRNA Expression in Glioma Cell Lines

Q-PCR was conducted to measure the expression levels of U251 and U87 cells. Total RNA was isolated using RNAiso Plus (9108, Takara). Total RNA (0.5–1 μg) was reverse-transcribed with miR224-3p, miR210 stem-loop and U6 snoRNA RT primers (RiboBio, Guangzhou, China) using a ReverTraAce qRT-PCR kit (FSQ-101, Toyobo) according to the manufacturer's protocol to synthesize cDNA. Real-time PCR was performed using a SYBR Premix Ex TaqTM Kit (QPK-201, Toyobo) with miR224-3p, miR210 and U6 snoRNA primers (miRQ0009198-1-1 for miR224-3p, miRQ0000267-1-1 for miR210, MQP-0201 for U6, RiboBio) as previously described [21 (link), 52 (link)]. The reactions were performed using a Lightcycler 2.0 instrument (Roche Applied Science). mRNA levels were normalized to GAPDH. U6 expression was used as the endogenous control for miRNA level. All data for each sample were collected in triplicate. The fold changes were calculated by relative quantification (2^−ΔΔCt). The primers used in the present study are listed in Supplementary Table S1.