MicroRNA profiling of low concentration extracellular vesicle RNA utilizing NanoString nCounter technology
Abstract Extracellular vesicles (EV) and the microRNAs that they contain are increasingly recognised as a rich source of informative biomarkers, reflecting pathological processes and fundamental biological pathways and responses. Their presence in biofluids makes them particularly attractive for bio...
| Main Authors: | , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Wiley
2023-01-01
|
| Series: | Journal of Extracellular Biology |
| Subjects: | |
| Online Access: | https://doi.org/10.1002/jex2.72 |
| _version_ | 1797766977045921792 |
|---|---|
| author | Rachel E. Crossland Anna Albiero Clara Sanjurjo‐Rodríguez Monica Reis Anastasia Resteu Amy E. Anderson Anne M. Dickinson Arthur G. Pratt Mark Birch Andrew W. McCaskie Elena Jones Xiao‐nong Wang |
| author_facet | Rachel E. Crossland Anna Albiero Clara Sanjurjo‐Rodríguez Monica Reis Anastasia Resteu Amy E. Anderson Anne M. Dickinson Arthur G. Pratt Mark Birch Andrew W. McCaskie Elena Jones Xiao‐nong Wang |
| author_sort | Rachel E. Crossland |
| collection | DOAJ |
| description | Abstract Extracellular vesicles (EV) and the microRNAs that they contain are increasingly recognised as a rich source of informative biomarkers, reflecting pathological processes and fundamental biological pathways and responses. Their presence in biofluids makes them particularly attractive for biomarker identification. However, a frequent caveat in relation to clinical studies is low abundance of EV RNA content. In this study, we used NanoString nCounter technology to assess the microRNA profiles of n = 64 EV low concentration RNA samples (180–49125 pg), isolated from serum and cell culture media using precipitation reagent or sequential ultracentrifugation. Data was subjected to robust quality control parameters based on three levels of limit of detection stringency, and differential microRNA expression analysis was performed between biological subgroups. We report that RNA concentrations > 100 times lower than the current NanoString recommendations can be successfully profiled using nCounter microRNA assays, demonstrating acceptable output ranges for imaging parameters, binding density, positive/negative controls, ligation controls and normalisation quality control. Furthermore, despite low levels of input RNA, high‐level differential expression analysis between biological subgroups identified microRNAs of biological relevance. Our results demonstrate that NanoString nCounter technology offers a sensitive approach for the detection and profiling of low abundance EV‐derived microRNA, and may provide a solution for research studies that focus on limited sample material. |
| first_indexed | 2024-03-12T20:33:04Z |
| format | Article |
| id | doaj.art-39d48e4470ce428d955614163bd77cde |
| institution | Directory Open Access Journal |
| issn | 2768-2811 |
| language | English |
| last_indexed | 2024-03-12T20:33:04Z |
| publishDate | 2023-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | Journal of Extracellular Biology |
| spelling | doaj.art-39d48e4470ce428d955614163bd77cde2023-08-01T18:55:33ZengWileyJournal of Extracellular Biology2768-28112023-01-0121n/an/a10.1002/jex2.72MicroRNA profiling of low concentration extracellular vesicle RNA utilizing NanoString nCounter technologyRachel E. Crossland0Anna Albiero1Clara Sanjurjo‐Rodríguez2Monica Reis3Anastasia Resteu4Amy E. Anderson5Anne M. Dickinson6Arthur G. Pratt7Mark Birch8Andrew W. McCaskie9Elena Jones10Xiao‐nong Wang11Translational and Clinical Research Institute, Faculty of Medical Sciences Newcastle University Newcastle upon Tyne UKDivision of Trauma and Orthopaedic Surgery, Department of Surgery University of Cambridge Addenbrooke's Hospital Cambridge UKTranslational and Clinical Research Institute, Faculty of Medical Sciences Newcastle University Newcastle upon Tyne UKTranslational and Clinical Research Institute, Faculty of Medical Sciences Newcastle University Newcastle upon Tyne UKTranslational and Clinical Research Institute, Faculty of Medical Sciences Newcastle University Newcastle upon Tyne UKTranslational and Clinical Research Institute, Faculty of Medical Sciences Newcastle University Newcastle upon Tyne UKTranslational and Clinical Research Institute, Faculty of Medical Sciences Newcastle University Newcastle upon Tyne UKTranslational and Clinical Research Institute, Faculty of Medical Sciences Newcastle University Newcastle upon Tyne UKDivision of Trauma and Orthopaedic Surgery, Department of Surgery University of Cambridge Addenbrooke's Hospital Cambridge UKDivision of Trauma and Orthopaedic Surgery, Department of Surgery University of Cambridge Addenbrooke's Hospital Cambridge UKLeeds Institute of Rheumatic and Musculoskeletal Medicine University of Leeds Leeds UKTranslational and Clinical Research Institute, Faculty of Medical Sciences Newcastle University Newcastle upon Tyne UKAbstract Extracellular vesicles (EV) and the microRNAs that they contain are increasingly recognised as a rich source of informative biomarkers, reflecting pathological processes and fundamental biological pathways and responses. Their presence in biofluids makes them particularly attractive for biomarker identification. However, a frequent caveat in relation to clinical studies is low abundance of EV RNA content. In this study, we used NanoString nCounter technology to assess the microRNA profiles of n = 64 EV low concentration RNA samples (180–49125 pg), isolated from serum and cell culture media using precipitation reagent or sequential ultracentrifugation. Data was subjected to robust quality control parameters based on three levels of limit of detection stringency, and differential microRNA expression analysis was performed between biological subgroups. We report that RNA concentrations > 100 times lower than the current NanoString recommendations can be successfully profiled using nCounter microRNA assays, demonstrating acceptable output ranges for imaging parameters, binding density, positive/negative controls, ligation controls and normalisation quality control. Furthermore, despite low levels of input RNA, high‐level differential expression analysis between biological subgroups identified microRNAs of biological relevance. Our results demonstrate that NanoString nCounter technology offers a sensitive approach for the detection and profiling of low abundance EV‐derived microRNA, and may provide a solution for research studies that focus on limited sample material.https://doi.org/10.1002/jex2.72extracellular vesiclemicroRNANanoStringprofiling |
| spellingShingle | Rachel E. Crossland Anna Albiero Clara Sanjurjo‐Rodríguez Monica Reis Anastasia Resteu Amy E. Anderson Anne M. Dickinson Arthur G. Pratt Mark Birch Andrew W. McCaskie Elena Jones Xiao‐nong Wang MicroRNA profiling of low concentration extracellular vesicle RNA utilizing NanoString nCounter technology Journal of Extracellular Biology extracellular vesicle microRNA NanoString profiling |
| title | MicroRNA profiling of low concentration extracellular vesicle RNA utilizing NanoString nCounter technology |
| title_full | MicroRNA profiling of low concentration extracellular vesicle RNA utilizing NanoString nCounter technology |
| title_fullStr | MicroRNA profiling of low concentration extracellular vesicle RNA utilizing NanoString nCounter technology |
| title_full_unstemmed | MicroRNA profiling of low concentration extracellular vesicle RNA utilizing NanoString nCounter technology |
| title_short | MicroRNA profiling of low concentration extracellular vesicle RNA utilizing NanoString nCounter technology |
| title_sort | microrna profiling of low concentration extracellular vesicle rna utilizing nanostring ncounter technology |
| topic | extracellular vesicle microRNA NanoString profiling |
| url | https://doi.org/10.1002/jex2.72 |
| work_keys_str_mv | AT rachelecrossland micrornaprofilingoflowconcentrationextracellularvesiclernautilizingnanostringncountertechnology AT annaalbiero micrornaprofilingoflowconcentrationextracellularvesiclernautilizingnanostringncountertechnology AT clarasanjurjorodriguez micrornaprofilingoflowconcentrationextracellularvesiclernautilizingnanostringncountertechnology AT monicareis micrornaprofilingoflowconcentrationextracellularvesiclernautilizingnanostringncountertechnology AT anastasiaresteu micrornaprofilingoflowconcentrationextracellularvesiclernautilizingnanostringncountertechnology AT amyeanderson micrornaprofilingoflowconcentrationextracellularvesiclernautilizingnanostringncountertechnology AT annemdickinson micrornaprofilingoflowconcentrationextracellularvesiclernautilizingnanostringncountertechnology AT arthurgpratt micrornaprofilingoflowconcentrationextracellularvesiclernautilizingnanostringncountertechnology AT markbirch micrornaprofilingoflowconcentrationextracellularvesiclernautilizingnanostringncountertechnology AT andrewwmccaskie micrornaprofilingoflowconcentrationextracellularvesiclernautilizingnanostringncountertechnology AT elenajones micrornaprofilingoflowconcentrationextracellularvesiclernautilizingnanostringncountertechnology AT xiaonongwang micrornaprofilingoflowconcentrationextracellularvesiclernautilizingnanostringncountertechnology |