HOME NEWS TECHNOLOGY SERVICES ABOUT US CONTACT US   Overview | Principle | Workflow | Analysis | Reproducibility | Robustness     FACTORIAL™ Technology: Overview Signals that control genome expression are executed through coordinated changes in the activities of transcription factors (TFs) - proteins that bind gene regulatory elements and modulate transcription. Mammalian genomes encode about 2000 TFs that, according to DNA binding similarities, can be classified into few hundred families. By analyzing activities of multiple TFs one should be able to obtain an informative snapshot of gene regulatory network. However, high-content analysis of TFs requires adequate tools. Of the few tools currently available to researchers who seek to study cell regulation at the level of transcription factor activities, none is geared towards quantitative high-content assessments. Assays that make use of reporter gene constructs (e.g., luciferase reporters) can be used to analyze only one or two transcription factors at a time. In contrast, DNA-binding assays can potentially be used to assess multiple transcription factors, but provide limited biological information because DNA binding is only one of many factors in the regulation of transcription factor activity. Attagene has conceptualized and developed a simple homogeneous assay, termed FactorialTM, enabling assessment of multiple transcription factors’ activities in a single experiment. With our FACTORIAL™ TF profilling system, you can... analyze basal and induced transcription factor activity profiles in a variety of commonly used cell lines assess the function of a gene of interest in the context of its effects on activities of multiple transcription factors investigate mechanisms of action of biologically active agents, including chemical compounds, peptides, siRNAs, and dominant-negative variants of proteins explore signal transduction pathways underlying alterations in gene expression profiles generated by microarray hybridization Back to the top Principle of the FACTORIAL™ Technology The crux of the FACTORIAL™ technology is a library of uniformly constructed Reporter Transcription Units (RTUs). Each FACTORIAL™ RTU is made in a common plasmid backbone and contains a unique TF-inducible promoter that is fused to a transcribed reporter sequence. When co-introduced into cell of interest, the RTUs produce reporter RNAs in amounts commensurable with the activities of the corresponding TFs present in a cell. To provide equal detection opportunities for different transcription factors, all FACTORIAL™ RTUs are supplied with essentially identical reporter sequences. To distinguish reporter sequences produced by different RTUs, each sequence is provided with a short processing tag (HpaI restriction cleavage site), position of which varies among the RTUs. Thus, reporter sequences can be discriminated upon cleavage with the corresponding processing enzyme. The cleaved reporter species are separated by high resolution capillary electrophoresis (sequencing) and quantified. A key aspect of the FACTORIAL™ technology is that all steps of the detection protocol are performed using a homogeneous set of reagents in a single reaction tube format, providing highly uniform conditions for detecting multiple transcription factors. Back to the top Workflow of FACTORIAL™ Assay For multiplexed detection of transcription factors the Factorial™ TF reporter library is introduced into cells of interest by using standard transfection procedure. Some time after transfection, total cellular RNA is isolated and reversely transcribed. The reporter cDNAs are amplified by PCR using a pair of primers that are common for all reporters. The PCR products are labeled with a fluorescent dye and digested by the HpaI. The digest produces a spectrum of fluorescently labeled DNA fragments of different lengths that are resolved by capillary electrophoresis (CE) and detected as separate fluorescent peaks. To perform the Factorial™ detection protocol you will need standard molecular biology equipment, including PCR thermocycler and capillary electrophoresis instrument (available from your nearest sequencing facility). Alternatively, you may submit your RNA samples to a designated Attagene's Factorial™ Detection facility. Back to the top FACTORIAL™ Assay Data Analysis The capillary electrophoresis data are processed by using Attagraph™; software. The software’ algorithms allow for subtraction of background fluorescence, precise sizing of the reporter peaks, and noise/reporter peaks discrimination. The relative fluorescent values of the individual reporter peaks as well as specific RTU activities values can be exported into Microsoft Office Excel for further analysis. The detection platform that ensures outstanding, reproducibility, and robustness that make FACTORIAL™ a truly quantitative assay. Back to the top FACTORIAL™ Reproducibility TF activity profile in HepG2 cells transiently transfected with the FACTORIAL™ library. Standardized FACTORIAL 30™ assay readout obtained by normalizing the signals of individual peaks to the sum of signals of all peaks. Means of data (± s.d.) obtained in 6 parallel experiments. Inset illustrates linearity (R2 1/2 0.9992) of signals generated by five calibrating TATA (black bars) that were present in the reporter library at defined concentrations. Back to the top FACTORIAL™ Robustness Two independent FACTORIAL 30™ experiments in HepG2 cells produced RNA that had apparently different degradation (inset). Each RNA sample was analyzed in triplicate by using FACTORIAL 30™ detection protocol, and data were calculated as described in Figure 2b. The bars represent mean values ±s.d. of the three assessments. Aliquots of one RNA sample generated during FACTORIAL 30™ assay in HepG2 cells were amplified by using variable numbers of PCR cycles. Each PCR condition was repeated in triplicate. The progress of amplification was monitored by quantitative real-time PCR performed in parallel (inset). The PCR products were collected after 27 cycles (exponential phase of PCR reaction) and after 40 cycles (saturated PCR) and further processed according to FACTORIAL 30™ detection protocol. Back to the top        ©Attagene,Inc; All rights reserved. LEGAL SITE MAP CUSTOMER SERVICE