Project description:Investigation in bacterial transcriptomics is widely used to investigate gene regulation, bacterial susceptibility to antibiotics, host-pathogen interactions, and pathogenesis. Transcriptomics is crucially dependent on suitable methods to isolate and detect bacterial RNA. Microfluidic approaches offer ways of creating integrated point-of-care systems, analysing a sample from preparation, RNA isolation through to detection. Critical for on-chip diagnostics to deliver on their promise is that mRNA expression is not altered through the use microfluidic sample processing. Here, we investigate the impact on the use of a microfluidic sample processing system based on hydrodynamic separation upon RNA expression of bacteria isolated from blood to prove its suitability for further microfluidic test development. A 10 array study using total RNA recovered from bacteria isolated using the microfluidic device and total RNA recovered from bacteria that were not separated using the device were compared. Arrays were performed in 5 biological replicates from each condition
Project description:Investigation in bacterial transcriptomics is widely used to investigate gene regulation, bacterial susceptibility to antibiotics, host-pathogen interactions, and pathogenesis. Transcriptomics is crucially dependent on suitable methods to isolate and detect bacterial RNA. Microfluidic approaches offer ways of creating integrated point-of-care systems, analysing a sample from preparation, RNA isolation through to detection. Critical for on-chip diagnostics to deliver on their promise is that mRNA expression is not altered through the use microfluidic sample processing. Here, we investigate the impact on the use of a microfluidic sample processing system based on hydrodynamic separation upon RNA expression of bacteria isolated from blood to prove its suitability for further microfluidic test development.
Project description:Fluorescence-activated cell sorting (FACS) is a specialized technique to isolate
cell subpopulations with a high level of recovery and accuracy. However, the cell sorting
procedure can impact the viability and metabolic state of cells. Here, we performed a comparative study and evaluated the impact of traditional high-pressure charged droplet-based and a microfluidic chip-based sorting approach on the metabolic and phosphoproteomic profile of different cell types. While microfluidic chip-based sorted cells more closely resembled the unsorted control group for most cell types tested, the droplet-based sorted cells showed significant metabolic and phosphoproteomic alterations. In particular, greater changes in redox and energy status were present in cells sorted with the droplet-based cell sorter along with higher transcriptional and spliceosomal regulation and mechanical stress signaling. These results
indicate microfluidic chip-based sorting is less disruptive compared to droplet-based sorting.
