Introduction

Historically, biological research in space has been constrained by the need to preserve samples and return them to Earth for analysis, a process that introduces significant delays and risks sample degradation. To overcome this limitation and enable more autonomous and timely research, NASA developed the WetLab-2 system, a suite of molecular biology tools designed for end-to-end gene expression analysis aboard the International Space Station (ISS). This publication details the successful validation of the WetLab-2 system, demonstrating for the first time that complex molecular procedures can be performed effectively in a microgravity environment.

Research Objective

The primary goal of this study was to validate the complete WetLab-2 workflow in microgravity. The specific objectives were to:

  • Demonstrate successful RNA isolation and purification from complex biological samples (bacterial cells and mammalian tissue) on the ISS.
  • Confirm that quantitative real-time PCR (RT-qPCR) functions reliably and efficiently in microgravity.
  • Compare the performance, accuracy, and data quality of the on-orbit system against identical 1g ground controls.

Key Findings

  • The WetLab-2 system successfully isolated high-quality RNA from both E. coli and mouse liver tissue aboard the ISS. On-orbit RNA quality (RIN 8.5) was comparable to ground controls (RIN 8.0).
  • On-orbit qPCR and RT-qPCR analysis was successful, with amplification efficiencies (104-108%) and cycle threshold (Ct) values closely matching ground controls (102-105%).
  • The entire workflow, from sample processing to data transmission to Earth, was completed in approximately 5 hours, enabling near real-time results.
  • A key microgravity-specific issue was identified: gas bubbles formed and were trapped in the optical window of the reaction tubes during thermal cycling, causing signal noise in the fluorescence data.
  • A follow-up experiment demonstrated that using standard, pressure-sealing commercial caps effectively suppressed bubble formation and produced smooth, high-quality amplification curves.
  • Lyophilized (freeze-dried) reagents were stable and performed comparably to standard wet lab reagents, reducing the need for limited on-orbit cold stowage.

Methodology

  • Organisms/Subjects: Frozen samples of Escherichia coli cells and 5 mg biopsies of mouse liver tissue were processed on the ISS.
  • Experimental Conditions: Experiments were performed by astronauts aboard the ISS in microgravity. Identical hardware and protocols were used to run parallel 1g control experiments on the ground within 24 hours.
  • Key Techniques: The study utilized the integrated WetLab-2 hardware suite, including a novel Sample Preparation Module (SPM) for RNA isolation, a bubble-removing pipette loader, and a Cepheid SmartCycler® for thermal cycling. Gene expression was analyzed using multiplex TaqMan-based Reverse Transcriptase-quantitative PCR (RT-qPCR).

Importance for Space Missions

This validation establishes a foundational capability for real-time molecular biology in space, which is critical for future long-duration missions to the Moon and Mars. The WetLab-2 system enables:

  • Rapid Health Diagnostics: Astronauts can quickly analyze biomarkers for stress, immune function, or infection, allowing for timely medical interventions.
  • Environmental Monitoring: The system can be used to identify microbial contaminants in the station’s air, water, or on surfaces, ensuring crew safety.
  • Iterative Science: Investigators can receive data from an experiment and use it to modify and conduct follow-up experiments while still on-orbit, dramatically accelerating the pace of scientific discovery.

Knowledge Gaps & Future Research

While a major success, this validation highlights areas for future work:

  • Confirming the long-term stability of lyophilized reagents under ambient (room temperature) storage conditions to further reduce reliance on cold stowage.
  • Adapting and validating the system for a wider range of biological samples, particularly human clinical samples like blood, saliva, and urine.
  • Integrating the RNA purification capability with other on-orbit technologies, such as nanopore sequencers, to enable comprehensive genomic and transcriptomic analysis.
  • Exploring opportunities to automate aspects of the workflow to further reduce demands on astronaut crew time.

Results

The successful validation of the WetLab-2 system marks a significant advancement for space-based biological research. By proving that complex molecular procedures like RNA extraction and RT-qPCR can be reliably performed in microgravity, this technology overcomes a major logistical barrier. It transitions space biology from a sample-return-dependent model to one that mirrors a dynamic, responsive Earth-based laboratory, paving the way for a deeper understanding of life in space and helping to ensure crew health on future deep-space exploration missions.

Data Visualization