小非编码 RNA 的拼接连接检测与标记

Detection and Labeling of Small Non-Coding RNAs by Splinted Ligation.

机构信息

Laboratoire CiTCoM "Cibles Thérapeutique et Conception des Médicaments", CNRS UMR 8038, Faculté de Pharmacie, Université de Paris, Paris, France.

Laboratoire de Biochimie, CNRS UMR 7654, Ecole Polytechnique, Palaiseau, France.

出版信息

Methods Mol Biol. 2021;2300:65-72. doi: 10.1007/978-1-0716-1386-3_7.

DOI:10.1007/978-1-0716-1386-3_7

PMID:33792872

Abstract

Discovery and characterization of microRNAs (miRNAs) and other families of small RNAs lead researchers to study their structures/functions and their expression patterns. The splinted ligation method described here is based on nucleic acid hybridization. It is optimized for the direct labeling and quantitative detection of small RNAs. A specific bridge DNA oligonucleotide is used, which is perfectly complementary to both the target small RNA and a labeled ligation nucleic acid. The target RNA is subsequently labeled by ligation, detected by analysis in denaturing conditions, and quantified by phosphorimaging. The protocol does not require any specific material, and the procedure is fast and sensitive.

摘要

发现和鉴定 microRNAs（miRNAs）和其他小 RNA 家族，促使研究人员研究它们的结构/功能和表达模式。本文描述的劈开连接法基于核酸杂交，经优化后可直接标记和定量检测小 RNA。使用的桥接 DNA 寡核苷酸与目标小 RNA 和标记连接核酸完全互补。随后通过连接使目标 RNA 被标记，在变性条件下通过分析进行检测，并通过磷显影进行定量。该方案不需要任何特定的材料，且操作快速灵敏。