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

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.

Nucleic acid extraction and sequencing from low-biomass synthetic Mars analog soils for in situ life detection

“…SETG operates by first extracting and isolating nucleic acids (DNA or RNA) from cells in solid or liquid samples using a modified Claremont BioSolutions (CBIO) solid-phase Purelyse® bacterial genomic DNA extraction kit. Prior studies have utilized OmniLyse, which is Purelyse® without extraction buffers, to lyse cells in an RNA extraction module aboard the International Space Station (Parra et al, 2017). Long-read sequencing is then conducted using the Oxford Nanopore Technologies (ONT) MinION which sequences nucleic acids via ionic current monitoring (Lu et al, 2016) and has been validated to sequence DNA in microgravity (Castro-Wallace et al, 2017;McIntyre et al, 2016), Lunar and Mars gravity (Carr/Zuber in prep 2018), and under simulated Mars temperature and pressure (Carr in prep 2018).…”

Fungal Biotechnology in Space: Why and How?

“…An extensive review on technologies in space for biological research is provided in Karouia et al (2017). In brief, high-throughput technologies such as MinION, miniPCR and WetLab-2 that enable DNA amplification (Boguraev et al 2017), RNA isolation and PCR analysis (Parra et al 2017) are available on board the ISS. WetLab-2 is a research platform for conducting real-time quantitative gene expression analysis aboard the ISS, allowing investigators to obtain real-time gene expression data from samples processed and analysed aboard the space station.…”

Diagnostics in space: will zero gravity add weight to new advances?

“…NASA has already achieved the first step in making routine nucleic acid isolation in space a reality with the successful development of WetLab-2 [8]. This system allows the analysis of gene expression to be conducted entirely onboard the ISS.…”

“…However, this single-use SPM is somewhat labor-intensive, and the crew can only process one sample at a time. Because of microgravity effects and the bubbles generated in the elution step, the collected eluate requires a procedure to remove the bubbles before being transferred to PCR reaction tubes for molecular analysis in the Cepheid SmartCycler real-time PCR thermal cycler (Table 1) onboard the ISS [8]. The next step is to improve upon this method with a less labor-intensive sample preparation method that reduces crew time and increases throughput.…”