We propose to acquire the QX200 AutoDG Droplet DigitalTM polymerase chain reaction (PCR) instrument to advance the basic and translational research of a large group of NIH-funded researchers at The George Washington University (GWU). The evaluation of limiting concentrations of RNA and DNA is often crucial for the detection and analysis of human diseases, for example the evaluation of RNA biomarkers to assess a patient's risk of coronary artery disease. The Droplet Digital PCR system provides absolute quantification of nucleic acid sequences with single copy resolution, enabling the researchers to overcome the current detection limits of conventional PCR systems. The increased sensitivity and precision of this system is achieved by partitioning the sample into 20,000 droplets and performance of the PCR reaction in each individual droplet. The Automatic Droplet Generator (AutoDG) reduces the variability between users and experiments and makes individual experiments highly reproducible. The acquisition of this piece of equipment will provide the necessary technology for the quantification of low copy numbers of RNA and DNA in patient samples or in vitro disease models, and permit the investigators the analysis of subtle RNA/DNA concentration changes that are associated with observable disease outcomes. Multiple NIH-funded research projects in the laboratories of 5 major and 6 minor users will benefit from the acquisition of this instrument, including the quantification of microRNA expression in infection-induced cancers, the characterization of the effects of HIV infection on the pathogenesis of atherosclerosis, the analysis of HIV eradication strategies in patient samples and humanized mice, the analysis of the role of cytokines in the effector CD8 T cell response against microsporidia, the characterization of the transcriptome of repetitive elements in T cells of HIV-infected patients, functional genomics for helminth parasite-induced cancers, the quantification of rare HIV cDNA, the characterization of gene expression changes and SNPs in parasitic hookworms, the analysis of mouse models of the dysphagia/22q11 deletion syndrome, the evaluation of RNA biomarkers for human coronary artery disease, and the analysis of epigenetic factors in HIV transcription and latency. The Droplet Digital PCR machine will be integrated into the existing instrument core of the GWU Department of Microbiology, Immunology and Tropical Medicine (MITM) and will be made available to collaborators within GWU and GWU-affiliated institutions in Washington D.C. MITM will manage the access and maintenance of the instrument and implement appropriate training programs for registered users. In conclusion, the proposed instrument acquisition will allow the investigators on this proposal as well as other potential future users to address research questions that they have so far been unable to answer due to technical limitations. This will provide a positive impact on NIH-funded research programs and create new collaborative venues for GWU's centers and institutions.
The requested Droplet Digital PCR (ddPCR) machine allows for the exact measurement of very low concentrations of DNA and RNA in patient samples and human disease models, which will enable the researchers at the George Washington University to better diagnose and analyze many human conditions whose disease progression is associated with subtle changes in nucleic acid concentrations. The ddPCR machine will advance several research projects that aim to understand the molecular and cellular basis of HIV/AIDS, cardiovascular diseases, neurological diseases, cancer, and other human conditions. The overall goal is to gain a deeper understanding of the mechanisms underlying these conditions and to develop new eradication strategies through basic and clinical research.
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| Thomas, Allison S; Jones, Kimberley L; Gandhi, Rajesh T et al. (2017) T-cell responses targeting HIV Nef uniquely correlate with infected cell frequencies after long-term antiretroviral therapy. PLoS Pathog 13:e1006629 |
| Raposo, Rui André Saraiva; de Mulder Rougvie, Miguel; Paquin-Proulx, Dominic et al. (2017) IFITM1 targets HIV-1 latently infected cells for antibody-dependent cytolysis. JCI Insight 2:e85811 |
| Jones, R Brad; Mueller, Stefanie; O'Connor, Rachel et al. (2016) A Subset of Latency-Reversing Agents Expose HIV-Infected Resting CD4+ T-Cells to Recognition by Cytotoxic T-Lymphocytes. PLoS Pathog 12:e1005545 |
