PDF(Pubmed)
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.
摘要：
无细胞RNA（cfRNA）是有希望的分析物作为非侵入性生物标志物，如果与代谢组学结合，则具有更大的潜力。血浆是cfRNA的最佳来源，但通常来自多种抗凝剂。在肝素中获得的血浆适用于代谢组学，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个简单的，省时，和具有成本效益的肝素酶方案，随后进行文库制备和人血浆cfRNAs的测序，并在-80°C的肝素中储存数年。在美国国立卫生研究院(NIH)临床中心的患有慢性HCV感染(NCT02400216)的患者的CPT™肝素钠管中收集血液。血浆cfRNA用肝素酶I处理并用于文库制备和下一代测序(NGS)。肝素酶处理维持了RNA完整性,并允许即使以7ngcfRNA作为起始材料的所有研究受试者的成功文库制备。源自PavianR包v1.2.0的分类报告显示没有人工读数。微生物读数上的脊索酸盐丰度表明通过肝素酶I处理没有增加实验误差。我们报告了一种新颖实用的方法，用于在肝素钠中收集和冷冻数年的人血浆的肝素酶治疗。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，然后可以与来自相同样本的代谢组学整合，最大限度地提高效率和减少抽血。
