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A Simple, Rapid, and Effective Heparinase Protocol to Enable Nucleic Acid Study from Frozen Heparinized Plasma
Cell-free RNAs (cfRNAs) are promising analytes as non-invasive biomarkers and have even greater potential if tied in with metabolomics. Plasma is an optimal source for cfRNAs but is often derived from a variety of anticoagulants. Plasma obtained in heparin is suitable for metabolomics but is difficu...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10660533/ https://www.ncbi.nlm.nih.gov/pubmed/37987359 http://dx.doi.org/10.3390/mps6060112 |
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author | Afruza, Rownock Minerva, Nicole Lack, Justin B. Chakraborty, Moumita Haddad, James A. Ali, Rabab O. Koh, Christopher Levy, Elliot B. Etzion, Ohad Heller, Theo |
author_facet | Afruza, Rownock Minerva, Nicole Lack, Justin B. Chakraborty, Moumita Haddad, James A. Ali, Rabab O. Koh, Christopher Levy, Elliot B. Etzion, Ohad Heller, Theo |
author_sort | Afruza, Rownock |
collection | PubMed |
description | Cell-free RNAs (cfRNAs) are promising analytes as non-invasive biomarkers and have even greater potential if tied in with metabolomics. Plasma is an optimal source for cfRNAs but is often derived from a variety of anticoagulants. Plasma obtained in heparin is suitable for metabolomics but is difficult to utilize for qPCR-based downstream analysis. In the present study, we aimed to develop a simple, time-efficient, and cost-effective heparinase protocol, followed by library preparation and sequencing of human plasma cfRNAs drawn and stored in heparin at −80 °C for several years. Blood was collected in CPT™ sodium heparin tubes from patients with chronic HCV infection (NCT02400216) at the National Institutes of Health (NIH) Clinical Center. Plasma cfRNAs were treated with heparinase I and used for library preparation and next-generation sequencing (NGS). Heparinase treatment maintained RNA integrity and allowed for successful library preparation for all the study subjects even with 7 ng of cfRNAs as starting material. The classification report derived from Pavian R package v1.2.0 showed no artificial reads. The abundance of chordate over microbial reads suggests no addition of experimental error through heparinase I treatment. We report a novel and practical approach to heparinase treatment for human plasma collected and frozen in sodium heparin for several years. This is an effective demonstration of utilizing heparin plasma for NGS and downstream transcriptomic research, which could then be integrated with metabolomics from the same samples, maximizing efficiency and minimizing blood draws. |
format | Online Article Text |
id | pubmed-10660533 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-106605332023-11-20 A Simple, Rapid, and Effective Heparinase Protocol to Enable Nucleic Acid Study from Frozen Heparinized Plasma Afruza, Rownock Minerva, Nicole Lack, Justin B. Chakraborty, Moumita Haddad, James A. Ali, Rabab O. Koh, Christopher Levy, Elliot B. Etzion, Ohad Heller, Theo Methods Protoc Protocol Cell-free RNAs (cfRNAs) are promising analytes as non-invasive biomarkers and have even greater potential if tied in with metabolomics. Plasma is an optimal source for cfRNAs but is often derived from a variety of anticoagulants. Plasma obtained in heparin is suitable for metabolomics but is difficult to utilize for qPCR-based downstream analysis. In the present study, we aimed to develop a simple, time-efficient, and cost-effective heparinase protocol, followed by library preparation and sequencing of human plasma cfRNAs drawn and stored in heparin at −80 °C for several years. Blood was collected in CPT™ sodium heparin tubes from patients with chronic HCV infection (NCT02400216) at the National Institutes of Health (NIH) Clinical Center. Plasma cfRNAs were treated with heparinase I and used for library preparation and next-generation sequencing (NGS). Heparinase treatment maintained RNA integrity and allowed for successful library preparation for all the study subjects even with 7 ng of cfRNAs as starting material. The classification report derived from Pavian R package v1.2.0 showed no artificial reads. The abundance of chordate over microbial reads suggests no addition of experimental error through heparinase I treatment. We report a novel and practical approach to heparinase treatment for human plasma collected and frozen in sodium heparin for several years. This is an effective demonstration of utilizing heparin plasma for NGS and downstream transcriptomic research, which could then be integrated with metabolomics from the same samples, maximizing efficiency and minimizing blood draws. MDPI 2023-11-20 /pmc/articles/PMC10660533/ /pubmed/37987359 http://dx.doi.org/10.3390/mps6060112 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Protocol Afruza, Rownock Minerva, Nicole Lack, Justin B. Chakraborty, Moumita Haddad, James A. Ali, Rabab O. Koh, Christopher Levy, Elliot B. Etzion, Ohad Heller, Theo A Simple, Rapid, and Effective Heparinase Protocol to Enable Nucleic Acid Study from Frozen Heparinized Plasma |
title | A Simple, Rapid, and Effective Heparinase Protocol to Enable Nucleic Acid Study from Frozen Heparinized Plasma |
title_full | A Simple, Rapid, and Effective Heparinase Protocol to Enable Nucleic Acid Study from Frozen Heparinized Plasma |
title_fullStr | A Simple, Rapid, and Effective Heparinase Protocol to Enable Nucleic Acid Study from Frozen Heparinized Plasma |
title_full_unstemmed | A Simple, Rapid, and Effective Heparinase Protocol to Enable Nucleic Acid Study from Frozen Heparinized Plasma |
title_short | A Simple, Rapid, and Effective Heparinase Protocol to Enable Nucleic Acid Study from Frozen Heparinized Plasma |
title_sort | simple, rapid, and effective heparinase protocol to enable nucleic acid study from frozen heparinized plasma |
topic | Protocol |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10660533/ https://www.ncbi.nlm.nih.gov/pubmed/37987359 http://dx.doi.org/10.3390/mps6060112 |
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