Quantitative Real-Time PCR Analysis

Quantitative real-time PCR (qRT-PCR) was performed as previously described 24 (link). Methods for mRNA expression detection were as follows: Briefly, first-strand cDNAs were synthesized using a mixture of oligo (dT)₁₅ and random primers with superscript reverse transcriptase (Invitrogen, Carlsbad, CA, USA). The absorption values of the SYBR Green I fluorescence in each tube were detected at the end of each thermal cycle. The housekeeping gene GAPDH was used as an internal control. Mature miR-16 level was detected using Bulge-Loop miRNA qRT-PCR kit (Ribobio). To normalize RNA content, the U6 snRNA was used as the internal control. PCR primers used in this study, as well as the size of fragments amplified, are shown in Table S2. Analyses were performed with a vii A7 Quantitative PCR System (Applied Biosystems, Carlsbad, CA, USA). Each sample was amplified in duplicate, and normalized versus the endogenous control. Results were calculated using the 2^−ΔΔCt method 25 (link).