A Simple, Rapid, and Effective Heparinase Protocol to Enable Nucleic Acid Study from Frozen Heparinized Plasma.

IF 2.3 Q3 BIOCHEMICAL RESEARCH METHODS Methods and Protocols Pub Date : 2023-11-20 DOI:10.3390/mps6060112
Rownock Afruza, Nicole Minerva, Justin B Lack, Moumita Chakraborty, James A Haddad, Rabab O Ali, Christopher Koh, Elliot B Levy, Ohad Etzion, Theo Heller
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Abstract

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.

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一个简单,快速,有效的肝素酶方案,使核酸研究从冷冻肝素化血浆。
无细胞rna (cfRNAs)是一种很有前途的非侵入性生物标志物,如果与代谢组学结合起来,它将具有更大的潜力。血浆是cfrna的最佳来源,但通常来源于多种抗凝血剂。从肝素中获得的血浆适合于代谢组学,但难以用于基于qpcr的下游分析。在本研究中,我们的目标是开发一种简单、高效、经济的肝素酶方案,然后对提取的人血浆cfrna进行文库制备和测序,并在-80°C的肝素中保存数年。从美国国立卫生研究院(NIH)临床中心的慢性HCV感染患者(NCT02400216)的CPT™钠肝素管中采集血液。血浆cfrna用肝素酶I处理,用于文库制备和下一代测序(NGS)。肝素酶处理保持了RNA的完整性,即使以7 ng的cfRNAs作为起始材料,也可以成功地为所有研究对象制备文库。从Pavian R包v1.2.0中导出的分类报告没有人为读取。在微生物读数中脊索动物的丰度表明通过肝素酶I处理没有增加实验误差。我们报告了一种新的实用的肝素酶治疗方法,用于收集并在肝素钠中冷冻数年的人血浆。这是利用肝素血浆进行NGS和下游转录组学研究的有效证明,然后可以将其与来自相同样品的代谢组学相结合,最大限度地提高效率并减少抽血。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Methods and Protocols
Methods and Protocols Biochemistry, Genetics and Molecular Biology-Biochemistry, Genetics and Molecular Biology (miscellaneous)
CiteScore
3.60
自引率
0.00%
发文量
85
审稿时长
8 weeks
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