用于小鼠显微注射实验的双链RNA的制备

Preparation of dsRNA for microinjection experiments in mouse.

作者信息

Svoboda Petr

机构信息

Institute of Molecular Genetics, Academy of Sciences of Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic.

出版信息

Cold Spring Harb Protoc. 2009 Jan;2009(1):pdb.prot5131. doi: 10.1101/pdb.prot5131.

DOI:10.1101/pdb.prot5131

PMID:20147026

Abstract

RNA interference (RNAi) is a suitable method for sequence-specific post-transcriptional gene silencing for a number of model systems. The protocol presented here is designed for the preparation of microgram amounts of double-stranded RNA (dsRNA) for microinjection experiments in mouse oocytes and early embryos. Briefly, dsRNA is produced after annealing sense and antisense RNA strands, or spontaneously during in vitro transcription of an inverted repeat. We usually include RNase T1 treatment of the annealed RNA prior to the purification step in order to remove unannealed single-stranded RNA (ssRNA), which can interfere with the quantification of dsRNA in a nondenaturing agarose gel.

摘要

RNA干扰（RNAi）对于许多模型系统而言，是一种适用于序列特异性转录后基因沉默的方法。此处介绍的方案旨在制备微克量的双链RNA（dsRNA），用于小鼠卵母细胞和早期胚胎的显微注射实验。简而言之，双链RNA是在正义链和反义链RNA退火后产生的，或者在反向重复序列的体外转录过程中自发产生。在纯化步骤之前，我们通常会对退火后的RNA进行RNase T¹处理，以去除未退火的单链RNA（ssRNA），因为它会干扰双链RNA在非变性琼脂糖凝胶中的定量分析。