Tuesday, October 4, 2011

Purification of polysome-engaged mRNA

Purification of polysome-engaged mRNA

mRNA is an excellent parameter of gene expression but it is not the only one. Thorough assessment of regulation of this aspect of the cellular biochemistry is multifaceted. Standard RNA isolation techniques, even when coupled to PCR, reveal information about the steady-state abundance of certain RNAs in the cell at the moment of cell lysis, yet reveal nothing at all about the translational fate of the transcripts of experimental interest. Recall that the gene expression is also controlled at the translational and posttranslational levels. Clearer definition of the translational aspect of gene expression may be gained by collecting and analyzing the mRNA fraction that has engaged the translational machinery. The polysome fraction of the cell (all mRNAs engaged by ribosomes) is a fairly accurate indicator of the proportion of the mRNA mass that has actually advanced to the translational level along the gene expression pathway. In the cell, some polysomes are associated with the endo- plasmic reticulum (presumably synthesizing secreted proteins, mitochondrial proteins, and proteins embedded in the membrane) while others remain as free polysomes, which are believed to synthesize proteins that will remain in the cytoplasm or move into the nucleus. Thus, the isolation of polysome-engaged mRNA is used to profile gene expression at the translational level and may well be a more accurate indicator of both efficiency of translation initiation as well as the phenotype identity of the cells under investigation.
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Bioelectronic DNA detection involves forming an electronic circuit mediated by nucleic acid hybridization and it serves as the basis for a DNA detection system called eSensor™ [1-4]. This system uses low-density DNA chips containing electrodes coated with DNA capture probes. Target DNA present in the sample hybridizes specifically both to capture probes and ferrocene labeled signal probes in solution thereby generating an electric current. Currente Sensor DNA chips contain as many as 36 electrodes for simultaneous detection of multiple pathogens from a single sample.

Many pathogens cause both acute and chronic disease at relatively low copy number and may be difficult or impossible to propagate in culture. Thus, most pathogen detection systems rely on nucleic acid amplification by using polymerase chain reaction (PCR). One highly effective amplification strategy targets conserved sequences among the family of organisms of interest. Such broad-range PCR strategies have been used to identify and characterize several known and previously uncharacterized bacteria [5,6] and viruses [7,8]. In order to maximize the utility of these effective pathogen nucleic acid amplification systems, amplification needs to be coupled with rapid, sensitive, and specific detection. Bioelectronic DNA detection by use of the eSensor chip might fulfill this need.

Human papillomaviruses (HPV) serve as an ideal model system for determining the efficiency and feasibility of eSensor DNA detection technology since there are at least 30 distinct genital HPV types that can be effectively amplified with broad-range consensus PCR primers. We designed two eSensor chips, each containing 14 probes specific for the conserved L1 region of the HPV genome. We evaluated clinical cervical cytology samples known to contain one or more HPV types. The eSensor DNA detection platform successfully detected the correct HPV type in most of these clinical samples, demonstrating that the system provides a rapid, sensitive, specific, and economical approach for multiple-pathogen detection and identification from a single sample.Background We used human papillomaviruses (HPV) as a model system to evaluate the utility of a nucleic acid, hybridization-based bioelectronic DNA detection platform (eSensor) in identifying multiple pathogens.