Pmir report mirna expression reporter vector system pmir

The 3′-UTR sequences of MCL-1 (GenBank accession number: NM_001197320) containing the putative miR-193b binding site were amplified by PCR using the cDNA from MCF-7/DOXR cells as a template. Primers for MCL-1 3′-UTR were as follows: forward, 5′-TACTGTAAGTGCAATAGT-3′; reverse, 5′-TACCATCTTCACTAAATCT-3′. PCR products were cloned into the pMIR-REPORT miRNA Expression Reporter Vector System (pMIR, Life Technologies, USA). The mutant plasmid was created by mutating the seed regions of the miR-193b binding sites using site-directed mutagenesis kit (Takara, Japan). MCL-1 expression vector was established for the “rescue” experiment, where the open reading frame of MCL-1 was cloned into pcDNA3.1 (Invitrogen, USA). The recombinant plasmid was named pcDNA3.1-MCL-1.

Total RNA was isolated by TRIzol. RNA was then reverse-transcribed into cDNA with M-MLV Reverse Transcriptase using the primer of oligo(dT) (Takara, Japan). The 3′-UTR region of human Mcl-1 (NM_001197320) was amplified by PCR using the cDNA as a template and cloned into the pMIR-REPORT miRNA Expression Reporter Vector System (pMIR, Life Technologies, USA). The recombinant plasmid was named pMIR-Mcl-1. The mutant plasmid was created by mutating the seed regions of the miR-26b-binding sites (UACUUGA to UAGAAGA) by using site-directed mutagenesis kit (Takara, Japan) and named pMIR-Mcl-1-M. The open reading frame of Mcl-1 gene without 3′-UTR was amplified by PCR with the cDNA as template and cloned into the pEGFP-N1 vector (Clontech, USA) and the resulting plasmid was named pEGFP-Mcl-1.