Human liver cdna

The VKOR coding sequence was PCR amplified from human liver cDNA (Clontech) using primers: VF1 (5′-CACCATGGGCAGCACCTGGGGGA-3′) and VR1 (5′-GCTCAGTGCCTCTTAGCCTT-3′) and TA-TOPO cloned into pcDNA3.1-V5/His (Invitrogen). The VKOR coding sequence was released by HindIII and NotI and then subcloned into the pcDNA3.1Hygro vector using the same restriction enzymes to give construct pVKORhygro (GenBank accession DM079693).

Total RNA of cultured cells was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Reverse transcription was performed with 400 ng RNA using random hexamer primers and Superscript II (Life Technologies). Human liver cDNA (Clontech, Mountain View, CA, USA) was used as positive control. RT-PCR (semi-nested) was arranged with different oligomer primers (DDC forward: 5-GCCCCTACTTCTTCGCCTAC-3; DDC forward nested: 5-ATGCTTGCGGACATGCTGTG-3; DDC reverse: 5-CAGTCTCCAGCTCTGTGCAT-3). PCR products were analyzed by using agarose gel electrophoresis with Midori Green (Nippon Genetics Europe GmbH, Düren, Germany) staining. All products were confirmed by sequencing [5 (link)].