Zaporozhchenko Ivan A, Morozkin Evgeniy S, Skvortsova Tatyana E, Bryzgunova Olga E, Bondar Anna A, Loseva Ekaterina M, Vlassov Valentin V, Laktionov Pavel P
Institute of Chemical Biology and Fundamental Medicine SB RAS (ICBFM SB RAS), Novosibirsk 630090, Russia.
Institute of Chemical Biology and Fundamental Medicine SB RAS (ICBFM SB RAS), Novosibirsk 630090, Russia.
Anal Biochem. 2015 Jun 15;479:43-7. doi: 10.1016/j.ab.2015.03.028. Epub 2015 Apr 2.
MicroRNAs (miRNAs) found in biological fluids such as blood and urine have been identified as promising biomarkers for many human disorders, including cancer, cardiopathies, and neurodegenerative diseases. However, circulating miRNAs are either encapsulated into vesicles or found in complexes with proteins and lipoproteins and, thus, require a special approach to their isolation. Acid phenol-chloroform extraction can solve this problem, but it is a labor-intensive procedure that relies heavily on the use of hazardous chemicals. Here we describe a fast and simple phenol-free protocol for miRNA isolation from biofluids. MiRNA is extracted from complexes with biopolymers by a high concentration of guanidine isothiocyanate combined with water/organic composition of solvents. Purification is finished using silica-based spin columns. Comparison of miRNA isolation from blood plasma and urine using the single-phase method and acid phenol-chloroform extraction by means of radioisotope spike-ins and quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) showed similar performance of the two methods.
在血液和尿液等生物体液中发现的微小RNA（miRNA）已被确定为许多人类疾病（包括癌症、心脏病和神经退行性疾病）的有前景的生物标志物。然而，循环miRNA要么被包裹在囊泡中，要么存在于与蛋白质和脂蛋白的复合物中，因此需要一种特殊的方法来进行分离。酸性酚-氯仿提取可以解决这个问题，但这是一个劳动密集型的过程，严重依赖于危险化学品的使用。在这里，我们描述了一种从生物体液中分离miRNA的快速简单的无酚方案。通过高浓度的异硫氰酸胍与水/有机溶剂组成相结合，从与生物聚合物的复合物中提取miRNA。使用基于硅胶的离心柱完成纯化。通过放射性同位素掺入和定量实时逆转录-聚合酶链反应（qRT-PCR），比较单相法和酸性酚-氯仿提取法从血浆和尿液中分离miRNA的效果，结果显示这两种方法具有相似的性能。
