植物微小RNA的分离与检测方法

Isolation and Detection Methods of Plant miRNAs.

作者信息

Vera-Hernández Pedro Fernando, de Folter Stefan, Rosas-Cárdenas Flor de Fátima

机构信息

Centro de Investigación en Biotecnología Aplicada del Instituto Politécnico Nacional (CIBA-IPN), Ex-Hacienda San Juan Molino, Tlaxcala, Mexico.

Unidad de Genómica Avanzada, Laboratorio Nacional de Genómica para la Biodiversidad (LANGEBIO), Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Irapuato, Guanajuato, Mexico.

出版信息

Methods Mol Biol. 2019;1932:109-120. doi: 10.1007/978-1-4939-9042-9_8.

DOI:10.1007/978-1-4939-9042-9_8

PMID:30701495

Abstract

Small RNAs (sRNAs) are RNAs of low abundance in organisms. Among sRNAs, miRNAs are included and represent approximately 10% of the total number of sRNAs. The isolation of sRNAs is critical for miRNA detection and analysis. The precipitation of low-molecular-weight (LMW) RNAs from total RNA extracts has allowed enrichment of sRNAs. Here, we describe a simple method to isolate sRNAs from different plant species. The main advantage of this method is that it does not need first an extraction of total RNA and it is not based on TRIzol reagent. This method has been successfully used for miRNA analyses by Northern blot assay and RT-qPCR (these techniques are as well described in this chapter), as well as sRNA library preparation.

摘要

小RNA（sRNA）是生物体中丰度较低的RNA。在sRNA中，包含微小RNA（miRNA），其约占sRNA总数的10%。sRNA的分离对于miRNA的检测和分析至关重要。从总RNA提取物中沉淀低分子量（LMW）RNA可实现sRNA的富集。在此，我们描述一种从不同植物物种中分离sRNA的简单方法。该方法的主要优点是无需首先提取总RNA，且不基于TRIzol试剂。此方法已成功用于通过Northern杂交分析和逆转录定量聚合酶链反应（RT-qPCR）（本章也对这些技术进行了描述）进行的miRNA分析以及sRNA文库制备。