一个简单,快速和有效的肝酶协议,使从冷的肝化血中进行核酸研究
Rownock Afruza1, Nicole Minerva1, Justin B Lack2
1Translational Hepatology Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Methods and protocols
|November 21, 2023
概括
一个新的肝酶协议使得从长期储存的肝化血中进行无细胞RNA (cfRNA) 分析. 这种方法保留了下一代测序的RNA完整性,促进了综合转录和代谢学研究.
科学领域:
- 生物标志物发现发现
- 分子生物学分子生物学
- 基因组学就是基因组学.
背景情况:
- 无细胞RNAs (cfRNAs) 是有价值的非侵入性生物标志物,有可能与代谢学集成.
- 血是cfRNAs的理想来源,但肝素抗凝剂会干扰标准的qPCR分析.
- 目前用于处理化血进行RNA分析的现有方法往往复杂或低效.
研究的目的:
- 为人类血cfRNAs开发一种简单,高效和具有成本效益的肝激酶协议.
- 为了实现从血中收集并存储在氨酸中多年的cfRNAs的下一代测序 (NGS).
- 证明肝素化血对综合转录和代谢学研究的有用性.
主要方法:
- 从慢性HCV感染患者的CPTTM肝素管中采集了血液样本.
- 血cfRNAs用肝酶I酶进行处理.
- 图书馆准备和下一代测序 (NGS) 在经过处理的cfRNAs上进行.
主要成果:
- 肝酶治疗成功地保持了RNA完整性,使得即使使用低起始材料 (7 ng cfRNAs) 也可以准备图书馆.
- NGS分析显示没有人造读数,并比微生物读数更多的带状细胞,这表明没有实验错误.
- 该方案在分析多年来在肝素中储存的血中的cfRNA方面被证明是有效的.
结论:
- 一种新且实用的肝酶治疗方法允许成功地对储存在肝中的人体血cfRNA进行NGS分析.
- 这种方法克服了氨酸抗凝剂对RNA分析的局限性,使得下游的转录基因研究成为可能.
- 使用化血用于NGS和转录组学最大限度地提高效率并最大限度地减少血液抽取,特别是在与代谢组学相结合时.
相关概念视频
01:30Anticoagulant Drugs: Low-Molecular-Weight Heparins
713
Hemostasis is a crucial process that prevents excessive blood loss from damaged blood vessels. It involves various mechanisms such as vasoconstriction, platelet adhesion and activation, and fibrin formation. The importance of each mechanism depends on the type of vessel injury. In contrast, thrombosis is the abnormal formation of a blood clot within the blood vessels, leading to potential complications if the clot obstructs blood flow. Thrombosis can be caused by increased coagulability of the...
713
01:24DNA Isolation
39.3K
DNA isolation protocols can be fast and straightforward or complex and time-consuming depending on the type and quality of DNA required for further processing. For example, plasmid DNA extraction is a bit more complicated than genomic DNA extraction because of the need for an appropriate lysis method to separate plasmid DNA from gDNA during isolation. However, for specific applications, such as long-range DNA sequencing that require a good yield of high- quality DNA samples, we need to follow...
39.3K