QIAamp UCP DNA Micro Kit

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The genomic DNA of MKN45 cells was isolated using a QIAamp UCP DNA Micro Kit (Qiagen, Inc., Valencia, CA, USA). The 3′-UTR sequence of the human E2F5 gene containing the miRNA-34a binding site was generated by polymerase chain reaction (PCR) with LA Taq DNA polymerase (Takara, Dalian, China) using a Eppendorf Mastercycler nexus Thermal Cycler (Eppendorf, Hamburg, Germany) with conditions as follows: Initial denaturation at 95°C for 5 min, followed by 33 cycles of 60 sec at 95°C for denaturing, 30 sec at 60°C for annealing and 60 sec at 72°C for extension, then a final extension at 72°C for 10 min. PCR was performed with the following primers: E2F5 UTR forward, CGG CTA GCT TCT GGA TTT CAA CTT TTC TTC; E2F5 UTR reverse, GCG TCG ACG GAA AGT GGA ATG TCA GAA GTC (Shanghai Shengong Biological Engineering Technology & Services, Ltd.). The 3′-UTR was subsequently cloned into the pmirGLO-report vector (Promega Corporation, Madison, WI, USA), which was denoted as pmirGLO-E2F5.



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