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

Autor:	Parra M; Space Biosciences Research Branch, NASA Ames Research Center, Moffett Field, California, United States of America., Jung J; Engineering Systems Division, NASA Ames Research Center, Moffett Field, California, United States of America.; KBRWyle, Mountain View, California, United States of America., Boone TD; Office of the Director, NASA Ames Research Center, Moffett Field, California, United States of America.; Millenium Engineering & Integration Co, Mountain View, California, United States of America., Tran L; Space Biosciences Research Branch, NASA Ames Research Center, Moffett Field, California, United States of America.; KBRWyle, Mountain View, California, United States of America., Blaber EA; Space Biosciences Research Branch, NASA Ames Research Center, Moffett Field, California, United States of America.; Universities Space Research Association, Mountain View, California, United States of America., Brown M; Applications Development, Claremont Biosolutions, Upland, California, United States of America., Chin M; Engineering Systems Division, NASA Ames Research Center, Moffett Field, California, United States of America.; Millenium Engineering & Integration Co, Mountain View, California, United States of America., Chinn T; Engineering Systems Division, NASA Ames Research Center, Moffett Field, California, United States of America.; Millenium Engineering & Integration Co, Mountain View, California, United States of America., Cohen J; Office of the Director, NASA Ames Research Center, Moffett Field, California, United States of America., Doebler R; Applications Development, Claremont Biosolutions, Upland, California, United States of America., Hoang D; Engineering Systems Division, NASA Ames Research Center, Moffett Field, California, United States of America.; Millenium Engineering & Integration Co, Mountain View, California, United States of America., Hyde E; Engineering Systems Division, NASA Ames Research Center, Moffett Field, California, United States of America.; Millenium Engineering & Integration Co, Mountain View, California, United States of America., Lera M; KBRWyle, Mountain View, California, United States of America.; Flight Systems Implementation Branch, NASA Ames Research Center, Moffett Field, California, United States of America., Luzod LT; Engineering Systems Division, NASA Ames Research Center, Moffett Field, California, United States of America., Mallinson M; Engineering Systems Division, NASA Ames Research Center, Moffett Field, California, United States of America., Marcu O; Space Biosciences Research Branch, NASA Ames Research Center, Moffett Field, California, United States of America.; KBRWyle, Mountain View, California, United States of America., Mohamedaly Y; Engineering Systems Division, NASA Ames Research Center, Moffett Field, California, United States of America.; Millenium Engineering & Integration Co, Mountain View, California, United States of America., Ricco AJ; Mission Design Division, NASA Ames Research Center, Moffett Field, California, United States of America.; Stanford University, Palo Alto, California, United States of America., Rubins K; NASA Astronaut Corps, NASA Johnson Space Center, Houston, Texas, United States of America., Sgarlato GD; Engineering Systems Division, NASA Ames Research Center, Moffett Field, California, United States of America.; KBRWyle, Mountain View, California, United States of America., Talavera RO; Engineering Systems Division, NASA Ames Research Center, Moffett Field, California, United States of America.; Millenium Engineering & Integration Co, Mountain View, California, United States of America., Tong P; Engineering Systems Division, NASA Ames Research Center, Moffett Field, California, United States of America.; Millenium Engineering & Integration Co, Mountain View, California, United States of America., Uribe E; Universities Space Research Association, Mountain View, California, United States of America., Williams J; NASA Astronaut Corps, NASA Johnson Space Center, Houston, Texas, United States of America., Wu D; KBRWyle, Mountain View, California, United States of America.; Mission Design Division, NASA Ames Research Center, Moffett Field, California, United States of America., Yousuf R; Space Biosciences Research Branch, NASA Ames Research Center, Moffett Field, California, United States of America.; KBRWyle, Mountain View, California, United States of America., Richey CS; Universities Space Research Association, Mountain View, California, United States of America., Schonfeld J; Engineering Systems Division, NASA Ames Research Center, Moffett Field, California, United States of America., Almeida EAC; Space Biosciences Research Branch, NASA Ames Research Center, Moffett Field, California, United States of America.
Jazyk:	angličtina
Zdroj:	PloS one [PLoS One] 2017 Sep 06; Vol. 12 (9), pp. e0183480. Date of Electronic Publication: 2017 Sep 06 (Print Publication: 2017).
DOI:	10.1371/journal.pone.0183480
Abstrakt:	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.
Databáze:	MEDLINE
Externí odkaz:	Zobrazit plný text záznamu Plný text ve formátu PDF