CryoGrid-PIXUL-RNA: high throughput RNA isolation platform for tissue transcript analysis.

Autor: Schactler SA; UW Medicine South Lake Union, University of Washington, Seattle, WA, 98109, USA.; Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, 98109, USA., Scheuerman SJ; UW Medicine South Lake Union, University of Washington, Seattle, WA, 98109, USA.; Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, 98109, USA., Lius A; UW Medicine South Lake Union, University of Washington, Seattle, WA, 98109, USA.; Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, 98109, USA., Altemeier WA; UW Medicine South Lake Union, University of Washington, Seattle, WA, 98109, USA.; Center for Lung Biology, University of Washington, Seattle, WA, 98109, USA., An D; UW Medicine South Lake Union, University of Washington, Seattle, WA, 98109, USA.; Center for Lung Biology, University of Washington, Seattle, WA, 98109, USA., Matula TJ; Center for Industrial and Medical Ultrasound, Applied Physics Laboratory, University of Washington, Seattle, WA, 98195, USA.; Matchstick Technologies, Inc, Kirkland, WA, 98033, USA., Mikula M; Department of Genetics, Maria Sklodowska-Curie National Research Institute of Oncology, 02-781, Warsaw, Poland., Kulecka M; Department of Genetics, Maria Sklodowska-Curie National Research Institute of Oncology, 02-781, Warsaw, Poland.; Department of Gastroenterology, Hepatology and Clinical Oncology, Centre for Postgraduate Medical Education, 01-813, Warsaw, Poland., Denisenko O; UW Medicine South Lake Union, University of Washington, Seattle, WA, 98109, USA., Mar D; UW Medicine South Lake Union, University of Washington, Seattle, WA, 98109, USA.; Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, 98109, USA., Bomsztyk K; UW Medicine South Lake Union, University of Washington, Seattle, WA, 98109, USA. karolb@uw.edu.; Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, 98109, USA. karolb@uw.edu.; Matchstick Technologies, Inc, Kirkland, WA, 98033, USA. karolb@uw.edu.
Jazyk: angličtina
Zdroj: BMC genomics [BMC Genomics] 2023 Aug 08; Vol. 24 (1), pp. 446. Date of Electronic Publication: 2023 Aug 08.
DOI: 10.1186/s12864-023-09527-7
Abstrakt: Background: Disease molecular complexity requires high throughput workflows to map disease pathways through analysis of vast tissue repositories. Great progress has been made in tissue multiomics analytical technologies. To match the high throughput of these advanced analytical platforms, we have previously developed a multipurpose 96-well microplate sonicator, PIXUL, that can be used in multiple workflows to extract analytes from cultured cells and tissue fragments for various downstream molecular assays. And yet, the sample preparation devices, such as PIXUL, along with the downstream multiomics analytical capabilities have not been fully exploited to interrogate tissues because storing and sampling of such biospecimens remain, in comparison, inefficient.
Results: To mitigate this tissue interrogation bottleneck, we have developed a low-cost user-friendly system, CryoGrid, to catalog, cryostore and sample tissue fragments. TRIzol is widely used to isolate RNA but it is labor-intensive, hazardous, requires fume-hoods, and is an expensive reagent. Columns are also commonly used to extract RNA but they involve many steps, are prone to human errors, and are also expensive. Both TRIzol and column protocols use test tubes. We developed a microplate PIXUL-based TRIzol-free and column-free RNA isolation protocol that uses a buffer containing proteinase K (PK buffer). We have integrated the CryoGrid system with PIXUL-based PK buffer, TRIzol, and PureLink column methods to isolate RNA for gene-specific qPCR and genome-wide transcript analyses. CryoGrid-PIXUL, when integrated with either PK buffer, TRIzol or PureLink column RNA isolation protocols, yielded similar transcript profiles in frozen organs (brain, heart, kidney and liver) from a mouse model of sepsis.
Conclusions: RNA isolation using the CryoGrid-PIXUL system combined with the 96-well microplate PK buffer method offers an inexpensive user-friendly high throughput workflow to study transcriptional responses in tissues in health and disease as well as in therapeutic interventions.
(© 2023. BioMed Central Ltd., part of Springer Nature.)
Databáze: MEDLINE
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