Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Afruza, Rownock, Minerva, Nicole, Lack, Justin B., Chakraborty, Moumita, Haddad, James A., Ali, Rabab O., Koh, Christopher, Levy, Elliot B., Etzion, Ohad, Heller, Theo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
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
_version_ 1785148412873670656
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
work_keys_str_mv AT afruzarownock asimplerapidandeffectiveheparinaseprotocoltoenablenucleicacidstudyfromfrozenheparinizedplasma
AT minervanicole asimplerapidandeffectiveheparinaseprotocoltoenablenucleicacidstudyfromfrozenheparinizedplasma
AT lackjustinb asimplerapidandeffectiveheparinaseprotocoltoenablenucleicacidstudyfromfrozenheparinizedplasma
AT chakrabortymoumita asimplerapidandeffectiveheparinaseprotocoltoenablenucleicacidstudyfromfrozenheparinizedplasma
AT haddadjamesa asimplerapidandeffectiveheparinaseprotocoltoenablenucleicacidstudyfromfrozenheparinizedplasma
AT alirababo asimplerapidandeffectiveheparinaseprotocoltoenablenucleicacidstudyfromfrozenheparinizedplasma
AT kohchristopher asimplerapidandeffectiveheparinaseprotocoltoenablenucleicacidstudyfromfrozenheparinizedplasma
AT levyelliotb asimplerapidandeffectiveheparinaseprotocoltoenablenucleicacidstudyfromfrozenheparinizedplasma
AT etzionohad asimplerapidandeffectiveheparinaseprotocoltoenablenucleicacidstudyfromfrozenheparinizedplasma
AT hellertheo asimplerapidandeffectiveheparinaseprotocoltoenablenucleicacidstudyfromfrozenheparinizedplasma
AT afruzarownock simplerapidandeffectiveheparinaseprotocoltoenablenucleicacidstudyfromfrozenheparinizedplasma
AT minervanicole simplerapidandeffectiveheparinaseprotocoltoenablenucleicacidstudyfromfrozenheparinizedplasma
AT lackjustinb simplerapidandeffectiveheparinaseprotocoltoenablenucleicacidstudyfromfrozenheparinizedplasma
AT chakrabortymoumita simplerapidandeffectiveheparinaseprotocoltoenablenucleicacidstudyfromfrozenheparinizedplasma
AT haddadjamesa simplerapidandeffectiveheparinaseprotocoltoenablenucleicacidstudyfromfrozenheparinizedplasma
AT alirababo simplerapidandeffectiveheparinaseprotocoltoenablenucleicacidstudyfromfrozenheparinizedplasma
AT kohchristopher simplerapidandeffectiveheparinaseprotocoltoenablenucleicacidstudyfromfrozenheparinizedplasma
AT levyelliotb simplerapidandeffectiveheparinaseprotocoltoenablenucleicacidstudyfromfrozenheparinizedplasma
AT etzionohad simplerapidandeffectiveheparinaseprotocoltoenablenucleicacidstudyfromfrozenheparinizedplasma
AT hellertheo simplerapidandeffectiveheparinaseprotocoltoenablenucleicacidstudyfromfrozenheparinizedplasma