title: RNA Extraction from a Mycobacterium under Ultrahigh Electric Field Intensity in a Microfluidic Device.
abstract: Studies of transcriptomes are critical for understanding gene expression. Release of RNA molecules from cells is typically the first step for transcriptomic analysis. Effective cell lysis approaches that completely release intracellular materials are in high demand especially for cells that are structurally robust. In this report, we demonstrate a microfluidic electric lysis device that is effective for mRNA extraction from mycobacteria that have hydrophobic and waxy cell walls. We used a packed bed of microscale silica beads to filter M. smegmatis out of the suspension. 4000-8000 V/cm field intensity was used to lyse M. smegmatis with long pulses (i.e., up to 30 pulses that were 5 s long each). Our quantitative reverse transcription (qRT)-PCR results showed that our method yielded a factor of 10-20 higher extraction efficiency than the current state-of-the-art method (bead beating). We conclude that our electric lysis technique is an effective approach for mRNA release from hard-to-lyse cells and highly compatible with microfluidic molecular assays.
authors.affiliation: Department of Biomedical Engineering and Mechanics, Virginia Tech , Blacksburg, Virginia 24061, United States.
authors.forename: Sai
authors.initials: S
authors.lastname: Ma
authors.affiliation: Department of Immunology and Infectious Diseases, Harvard School of Public Health , Boston, Massachusetts 02115, United States.
authors.forename: Bryan D
authors.initials: BD
authors.lastname: Bryson
authors.affiliation: Department of Biomedical Engineering and Mechanics, Virginia Tech , Blacksburg, Virginia 24061, United States.
authors.forename: Chen
authors.initials: C
authors.lastname: Sun
authors.affiliation: Department of Immunology and Infectious Diseases, Harvard School of Public Health , Boston, Massachusetts 02115, United States.
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Fortune
authors.affiliation: Department of Chemical Engineering, Virginia Tech , Blacksburg, Virginia 24061, United States.
authors.forename: Chang
authors.initials: C
authors.lastname: Lu
mesh_terms: D004574:Electromagnetic Fields; D056656:Lab-On-A-Chip Devices; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D020102:Mycobacterium smegmatis / Q000235:genetics; D012313:RNA / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012822:Silicon Dioxide / Q000737:chemistry
chemical_list: D012313:RNA; D012822:Silicon Dioxide