BMC Genomics 2005, 6:88 http://www.biomedcentral.com/1471-2164/6/88 The control plasmid for monitoring transfection effi- ciency is MT-RLuc [40]. miRNA plasmids were constructed by cloning a DNA seg- ment containing the predicted primary transcript of each miRNA into the BamHI/EcoRI site of ActMSI [40]. Each primary transcript was amplified by PCR from genomic DNA to generate fragments with the following sequence coordinates: let-7: 2L:18450072-18450291; mir-92b: 3R:21466427-21466673; mir-312: 2R:15647675- 15647897; mir-34: 3R:5926642-5926792. In each case, BamHI and EcoRI sites were introduced at the 5' and 3' ends, respectively, by PCR. Targeting Assay S2 cells were transfected with 2 µ g of microRNA plasmid (for non-control samples), 50 ng reporter plasmid, and 10 ng control plasmid. For each transfection, 0.5 mL Schnei- der's Drosophila Medium (Gibco) containing indicated plasmids and 0.5 mL Schneider's Drosophila Medium containing 5 µ L Cellfectin (Invitrogen) were mixed gently and incubated at RT for 15–45 minutes. 2 × 106 cells were centrifuged for 5 minutes at 1000 g, aspirated, resus- pended in the DNA-lipid mix described above, and trans- ferred to a 35 mm well of a 6-well plate. Cells were incubated at 25°C for the remainder of the transfection. After 4–5 hours, 0.5 mL of Schneider's Drosophila Medium containing 30% Fetal Bovine Serum (Gibco) was added to each well. The following day, 2 mL of complete growth medium [Schneider's Drosophila Medium with 10% FBS and 1% Pen/Strep (Gibco)] was added to each well. Between 38 and 45 hours after transfection, the reporter and control plasmids were induced by adding 3.5 µ L of 700 mM CuSO4. After 6–6.5 hrs, cells were har- vested, lysed, and assayed for reporter and control luci- ferase expression using the Dual-Luciferase Reporter Assay system (Promega). Each transfection was carried out at least 5 times. Potential miRNA Target Database D. melanogaster 3'UTRs were obtained from the Berkeley Drosophila Genome Project http://www.fruitfly.org, Dro- sophila Release 3.1 Annotations. Conserved D. pseudoo- bscura sequences were obtained from the Berkeley Genome Pipeline using the following parameters: mini- mum conservation width = 1; calculation window = 20; minimum conservation = 80%. We used the 7-8-03 AVID alignment for determining conserved regions [30,31] miRNA sequences D. melanogaster miRNA sequences were obtained from The microRNA Registry [6]. Results In this initial use of MovingTargets we set stringent values for all of the adjustable biological constraints to produce a high likelihood set of miRNA target predictions. The fol- lowing values were used: minimum of 3 target sites; max- imum free energy of hybridization of -15 kcal/mole at room temperature (22°C) for each target site; minimum 7 out of 8 consecutive 5' miRNA nt matches; maximum of 1 G:U base pair in the miRNA 5' region. The high-likeli- hood set of miRNA target predictions corresponding to these strict biological constraints, generated from analysis of all 78 miRNAs and the full database of 6399 potential targets containing conserved 3' UTR sequences, is given in Table 1. Given the strict constraints, this group will not contain all miRNA/target pairs (and includes no predic- tions for a subset of the known miRNAs). A striking feature of the set of predicted miRNA targets is the disproportionate fraction of mRNAs that encode tran- scription factors (13 of 41 genes with known function) or have assigned roles in neural processes (7 of 41). [41] noted a similar enrichment for transcription factors and neural genes in predicted miRNA targets in Drosophila. Because the different predictions identify groups of genes that are not fully overlapping, the bias is even more strik- ing. The emphasis on transcription factors was also observed for predicted miRNA targets in plants [42] and mammalian cells [43]. Validation of predicted targets We chose a subset of three predicted miRNA-target pairs for validation in a cultured cell assay. The assay is similar to that used by Zeng and Cullen [22]. A reporter plasmid expresses, under control of the inducible metallothionein promoter [44], a hybrid mRNA in which the firefly luci- ferase coding region is fused to the candidate target 3' UTR (luc/target). The miRNA plasmid expresses, under control of the constitutive actin promoter, a genomic DNA seg- ment that contains the miRNA primary transcript. Finally, a control plasmid expresses Renilla luciferase under con- trol of the inducible metallothionein promoter, and serves to monitor transfection efficiency. In parallel trans- fections one population of S2 cells receives all of the plas- mids, while a second population receives the reporter and control, but not the miRNA plasmid. Approximately 1.5 days after transfection (to allow the miRNA to accumu- late), transcription of the reporter and control mRNA is induced by addition of CuSO4 to the growth medium. After an additional 6 hours, the cells are harvested and the levels of firefly and Renilla luciferase are measured. The let-7 miRNA is predicted to target the abrupt (ab) mRNA at five positions in the 3' UTR (Table 1, Fig. 1A). The level of luciferase activity from the luc/ab mRNA in the absence of exogenous miRNA provides the standard for Page 4 of 11 (page number not for citation purposes)