Total RNA was extracted using the TRIzol reagent (Invitrogen, 15596018) as described in the manufacturer′s instructions, and cDNAs were synthesized with the SuperScript™ First-Strand Synthesis system (Invitrogen, 18091200). The target mRNA content in cells was measured by fluorescence quantitative PCR. The primers used in this study were as follows: human BECN1 (forward, 5′-TCA CCA TCC AGG AAC TCA C-3′; reverse, 5′-GGA TCA GCC TCT CCT CCT CT-3′), and human GAPDH (forward, 5′-GAC TCA TGA CCA CAG TCC ATG C-3′; reverse, 5′-CAG GTC AGG TCC ACC ACT GA-3′).
For miRNA determination, total miRNAs were extracted from cells using the miRNeasy Mini Kit (Qiagen, 218161), and miRNA expression was evaluated by using a Q6 real-time PCR system (Applied Biosystems) and the miScript PCR Kit (Qiagen). The primer for MIR516A (5′-TGC TTC CTT TCA GAG GGT-3′) was synthesized by Sunny Biotechnology, and RNU6 was used as an internal loading control with the primer provided in the miScript PCR Kit. Data were analyzed as described in a previous publication [60 (link)].
For miRNA determination, total miRNAs were extracted from cells using the miRNeasy Mini Kit (Qiagen, 218161), and miRNA expression was evaluated by using a Q6 real-time PCR system (Applied Biosystems) and the miScript PCR Kit (Qiagen). The primer for MIR516A (5′-TGC TTC CTT TCA GAG GGT-3′) was synthesized by Sunny Biotechnology, and RNU6 was used as an internal loading control with the primer provided in the miScript PCR Kit. Data were analyzed as described in a previous publication [60 (link)].
