Microgravity validation of a novel system for RNA isolation and multiplex quantitative real time PCR analysis of gene expression on the International Space Station

PLoS One. 2017 Sep 6;12(9):e0183480. doi: 10.1371/journal.pone.0183480. eCollection 2017.

Abstract

The International Space Station (ISS) National Laboratory is dedicated to studying the effects of space on life and physical systems, and to developing new science and technologies for space exploration. A key aspect of achieving these goals is to operate the ISS National Lab more like an Earth-based laboratory, conducting complex end-to-end experimentation, not limited to simple microgravity exposure. Towards that end NASA developed a novel suite of molecular biology laboratory tools, reagents, and methods, named WetLab-2, uniquely designed to operate in microgravity, and to process biological samples for real-time gene expression analysis on-orbit. This includes a novel fluidic RNA Sample Preparation Module and fluid transfer devices, all-in-one lyophilized PCR assays, centrifuge, and a real-time PCR thermal cycler. Here we describe the results from the WetLab-2 validation experiments conducted in microgravity during ISS increment 47/SPX-8. Specifically, quantitative PCR was performed on a concentration series of DNA calibration standards, and Reverse Transcriptase-quantitative PCR was conducted on RNA extracted and purified on-orbit from frozen Escherichia coli and mouse liver tissue. Cycle threshold (Ct) values and PCR efficiencies obtained on-orbit from DNA standards were similar to Earth (1 g) controls. Also, on-orbit multiplex analysis of gene expression from bacterial cells and mammalian tissue RNA samples was successfully conducted in about 3 h, with data transmitted within 2 h of experiment completion. Thermal cycling in microgravity resulted in the trapping of gas bubbles inside septa cap assay tubes, causing small but measurable increases in Ct curve noise and variability. Bubble formation was successfully suppressed in a rapid follow-up on-orbit experiment using standard caps to pressurize PCR tubes and reduce gas release during heating cycles. The WetLab-2 facility now provides a novel operational on-orbit research capability for molecular biology and demonstrates the feasibility of more complex wet bench experiments in the ISS National Lab environment.

Publication types

  • Validation Study

MeSH terms

  • Animals
  • Escherichia coli / genetics
  • Freeze Drying
  • Gene Expression Regulation*
  • Liver / metabolism
  • Mice
  • Multiplex Polymerase Chain Reaction / methods*
  • RNA / genetics
  • RNA / isolation & purification*
  • Real-Time Polymerase Chain Reaction / methods*
  • Reproducibility of Results
  • Spacecraft*
  • Weightlessness*

Substances

  • RNA

Grant support

Funding for this research was provided by the National Aeronautics and Space Administration’s International Space Station (ISS) Program at Johnson Space Center for development of a microgravity RT-qPCR analysis facility. The project was independently conducted by NASA Ames Research Center Space Biosciences and Engineering Systems Divisions. The NASA ISS funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.