姚媛,陈悟,陈曦,韩逸群,朱彤.环境流行病学研究中全血长期保存与RNA提取方法[J].环境科学学报,2019,39(12):4301-4308
环境流行病学研究中全血长期保存与RNA提取方法
- Long-term storage of whole blood samples and RNA isolation in environmental epidemiological studies
- 基金项目：国家自然科学基金（No.81571130100，41421064，21190051，41121004）；国家重点基础研究发展（973）计划（No.2015CB553401）
- 姚媛
- 北京大学工程科学与新兴技术高精尖创新中心, 北京大学环境科学与工程学院环境模拟与污染控制国家重点联合实验室, 北京 100871
- 陈悟
- 北京大学工程科学与新兴技术高精尖创新中心, 北京大学环境科学与工程学院环境模拟与污染控制国家重点联合实验室, 北京 100871
- 陈曦
- 1. 北京大学工程科学与新兴技术高精尖创新中心, 北京大学环境科学与工程学院环境模拟与污染控制国家重点联合实验室, 北京 100871;2. 深圳市建筑科学研究院股份有限公司, 研创中心, 深圳 518049
- 韩逸群
- 1. 北京大学工程科学与新兴技术高精尖创新中心, 北京大学环境科学与工程学院环境模拟与污染控制国家重点联合实验室, 北京 100871;2. MRC-PHE Centre for Environment and Health, School of Population Health & Environmental Sciences, King's College London, London SE1 9NH
- 朱彤
- 北京大学工程科学与新兴技术高精尖创新中心, 北京大学环境科学与工程学院环境模拟与污染控制国家重点联合实验室, 北京 100871
- 摘要：RNA是在基因表达层面探究分子生物学机制的重要研究对象.由于RNA化学性质活泼、结构不稳定，并且内外源RNA酶无处不在，如何从环境流行病学研究收集到的全血样本中提取出高质量的总RNA是困扰研究人员的一大难题.本研究旨在建立一种能长期保存全血样本并从中提取出高质量总RNA的方法.通过将环境流行病学研究中采集到的少量新鲜全血立即与10倍体积的TRIzol试剂混匀后置于-80℃冰箱中，可保护RNA在长期保存过程中不发生降解.针对长期保存的全血-TRIzol混合体系，在经典的TRIzol一步法基础上，无需将全血样本分装至不含RNA酶的1.5 mL离心管，直接在较大操作空间（不含RNA酶的15 mL离心管）中进行总RNA相分离、沉淀、洗涤等操作，并在总RNA晾干前再增加离心并吸取少量残留液体操作，缩短自然晾干时间.本方法无需离心、分离血浆、裂解红细胞等繁杂的前处理步骤，极大地简化了操作步骤，减少了采样工作量，实现了对全血样本的低成本长期保存.通过增大操作空间、改进晾干操作，本方法显著提高了总RNA样本的完整性和纯度.使用本方法提取出保存一年的全血总RNA产率为（9.18±2.10）μg·mL-1，完整性指标RIN值和28S/18S分别为8.98±0.18和1.08±0.08，纯度指标OD260/OD280和OD260/OD230分别为2.18±0.04和1.70±0.00.保存两年的全血提取出总RNA的产率、完整性及纯度仍然可以满足下游实验要求.
- Abstract：RNA is an important research object to explore the molecular biological mechanism at the level of gene expression. Due to the unstable structure and active chemical property of RNA and the influence of endogenous and exogenous RNase, how to isolate high-quality total RNA from whole blood samples in environmental epidemiological studies is a critical issue. The purpose of this study is to establish a method for long-term storage of whole blood samples and isolation of high-quality total RNA. A small amount of fresh whole blood was immediately mixed with 10 times the volume of TRIzol reagent and stored in a -80℃ refrigerator to protect RNA from degradation in long-term storage. Based on the conventional one-step method, instead of aliquoting the whole blood-TRIzol mixture into 1.5 mL RNase-free centrifuge tubes, total RNA phase separation, precipitation and washing were performed directly in the original mixture. Additionally, centrifugation and discarding residual liquid before total RNA drying helped shorten the natural drying time. This method eliminated the need for centrifugation, separation of plasma, lysis of red blood cells and other time-consuming pretreatment steps, and achieved low-cost and long-term storage of whole blood samples. This method significantly improved the integrity and purity of the total RNA by increasing the operating space (15 mL RNase-free centrifuge tubes) and improving the drying procedure. The results showed that for whole blood stored for one year, the yield of total RNA was (9.18±2.10) μg·mL-1, the RIN value and 28S/18S were 8.98±0.18 and 1.08±0.08, respectively, and OD260/OD280 and OD260/OD230 were 2.18±0.04 and 1.70±0.00, respectively. The yield, integrity and purity of the total RNA isolated from whole blood stored for two years could still meet the requirements of downstream experiments.
