Takane et al. BMC Genomics 2010, 11:101 http://www.biomedcentral.com/1471-2164/11/101 as orthologous genes conserved in at least four species, each containing the miRNA target site of interest. Expression vectors To construct target-site reporter plasmids, each DNA fragment (3’-UTR sequence of the DLL1 gene (668 nt; accession no. AF003522), ELK3 gene (519 nt; accession no. BC017371), EIF2C4 gene (2148 nt; accession no. AB046787), TAGLN2 gene (1391 nt; accession no. D21261), LARP4 gene (1678 nt; accession no. AY004310), CNN3 gene (1391 nt; accession no. BC025372), and ATP6V1B2 gene (1208 nt; accession no. L35249)) was amplified from HeLa genomic DNA via polymerase chain reaction using site-specific primers and was inserted into the XhoI/NotI sites of the psi- CHECK-2 plasmid vector (which encodes both firefly and Renilla luciferases; Promega, Madison, WI, USA) (Figure 4A). The oligonucleotide was designed to intro- duce XhoI and NotI sites at the 5’ and 3’ termini, respectively. The resulting plasmids were termed pLuc- DLL1, pLuc-CNN3, pLuc-LARP4, pLuc-ELK3, pLuc- EIF2C4, pLuc-TAGLN2, and pLuc-ATP6V1B2, respec- tively. miRIDIAN™ miRNA Mimic for hsa-let-7a, hsa- miR-1, hsa-miR-124, hsa-miR-34a, and negative control (miRIDIAN microRNA Hairpin Inhibitor Negative Con- trol #1) were purchased from Dharmacon. miRNA Mimic molecules are chemically modified double- stranded RNA oligonucleotides. The sequences of the oligodeoxyribonucleotides used for PCR were as follows: (1) DLL1_S: 5’-TTACTCGAGAATGGAAGTGA GATGGCAAGAC-3’ (2) DLLl_A: 5’-TTAGCGGCCGCTTGTCATTCA TAAAATTTATTT-3’ (3) CNN3_S: 5’-TTACTCGAGTCCACACAGAAG GAGCTCAG-3’ (4) CNN3_A: 5’-TTAGCGGCCGCCAGGAAGAG CAAATGCATCA-3’ (5) LARP4_S: 5’-TTACTCGAGGTTCCCATTT GATGGCATGT-3’ (6) LARP4_A: 5’-TTAGCGGCCGCCATAG CACCTTGGCGATGTT-3’ (7) ELK3_S: 5’-TTACTCGAGCGTCTGGCCACAAT TAAGGA-3’ (8) ELK3_A: 5’-TTAGCGGCCGCTGCTTTCA TATTGCCCACTG-3’ (9) EIF2C4_S: 5’-TTACTCGAGTTCTACCAG CAGCTCGGAAT-3’ (10) EIF2C4_A: 5’-TTAGCGGCCGCTTGGATTC CAGCAAGTCCTC-3’ (11) TAGLN2_S: 5’-TTACTCGAGCCCTCCCAC GAATGGTTAAT-3’ (12) TAGLN2_A: 5’-TTAGCGGCCGCATGGAAAAT GAGAAGCCACG-3’ Page 11 of 13 (13) ATP6V1B2_S: 5’-TTACTCGAGTCCGCGCTCTT GTGAAATAC-3’ (14) ATP6V1B2_A: 5’-TTAGCGGCCGCATAAT CATGCTGACTCCCCC-3’ Transfection and luciferase reporter assay Transient transfection and luciferase assays were per- formed as described previously, with slight modifications [22]. Briefly, HeLa cells were grown in 10% FBS in DMEM and seeded in 24-well plates 24 h before trans- fection. Cells were transfected with the indicated amounts of reporter and miRNA Mimic (100 ng of tar- get reporter and 5, 20, and 60 pmol of miRNA Mimic) in the presence of Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Firefly and Renilla luciferase activ- ities were measured consecutively using the Dual-luci- ferase assay system (Promega) 24 h after transfection, according to the manufacturer’s instructions. List of abbreviations used miRNA: microRNA; 3’-UTR: 3’-untranslated region; EC value: multiplied value of Enrichment and Coverage; nt: nucleotides; SD: standard deviation. Additional file 1: Basic concept of miRNA/mRNA duplex formation. An example of the binding pattern of miRNA (bottom)/mRNA (top) duplexes is shown using cel-let-7 and die-1 3’-UTR sequences from C. elegans. The green and blue squares depict mismatched nucleotide sequences of the mRNA and miRNA, respectively. The red square depicts G-U wobble pairs within the whole miRNA sequence and the black arrow pinpoints a G-U wobble pair within the seed sequence. Click here for file [ http://www.biomedcentral.com/content/supplementary/1471-2164-11- 101-S1.PDF ] Additional file 2: Summary of the number of target genes in each extraction step Click here for file [ http://www.biomedcentral.com/content/supplementary/1471-2164-11- 101-S2.PDF ] Additional file 3: Additional examples of miRNA target sites in orthologous gene transcripts used for experimental verification. Potential target sites of miR-124 (orange arrows) in the 3’-UTR sequences of orthologous ELK3 transcripts (A). Potential target sites of let-7 (green arrows) in the 3’-UTR sequences of orthologous EIF2C4 transcripts (B). Potential target sites of miR-1 (blue arrows) in the 3’-UTR sequences of orthologous TAGLN2 transcripts (C) and ATP6V1B2 transcripts (D). 3’-UTR sequences and miRNAs are shown in dotted boxes for each potential target site; the colours of dotted boxes and arrows correspond to those of each miRNA. Click here for file [ http://www.biomedcentral.com/content/supplementary/1471-2164-11- 101-S3.PDF ] Acknowledgements We are grateful to Mizuki Komasa, Motomu Matsui, and Hikaru Taniguchi (Keio University, Japan) for useful discussions. This research was supported in part by a Grant-in-Aid from the 21st Century Centre of Excellence (COE) Program entitled “Understanding and Control of Life’s Function via Systems Biology” (Keio University); research funds from the Yamagata Prefectural Government and Tsuruoka City in Japan; and a grant from Keio University.