High-resolution northern blot for a reliable analysis of microRNAs and their precursors.
High-resolution northern blot for a reliable analysis of microRNAs and their precursors.
复制标题
DOI：
10.1100/tsw.2011.11
复制
发表时间：
2011-01-05
影响因子：
--
通讯作者：
Krzyzosiak WJ
中科院分区：
文献类型：
--
作者：
Koscianska E;Starega-Roslan J;Czubala K;Krzyzosiak WJ
该协议描述了如何进行Northern杂交分析，以单核苷酸分辨率检测microRNAs及其前体，这对于分析单个长度变异和评估细胞中独特的microRNAs的相对数量至关重要。Northern印迹分析包括通过凝胶电泳法解析RNA，然后转移并固定到尼龙膜上，以及通过与放射性探针杂交进行检测。早期改进这种方法的努力主要集中在改变短RNA检测的灵敏度上。通过优化电泳步骤，提高了Northern印迹技术的分辨率。我们还研究了该程序的其他步骤，目的是提高RNA的分辨率；在此，我们提出了几项建议。我们的方法适用于所有类型的内源和外源RNA的分析，长度范围分别为20-30和50-70 NT，分别对应于microRNA和前microRNA的长度。
This protocol describes how to perform northern blot analyses to detect microRNAs and their precursors with single-nucleotide resolution, which is crucial for analyzing individual length variants and for evaluating relative quantities of unique microRNAs in cells. Northern blot analysis consists of resolving RNAs by gel electrophoresis, followed by transferring and fixing to nylon membranes as well as detecting by hybridization with radioactive probes. Earlier efforts to improve this method focused mainly on altering the sensitivity of short RNA detection. We have enhanced the resolution of the northern blot technique by optimizing the electrophoresis step. We have also investigated other steps of the procedure with the goal of enhancing the resolution of RNAs; herein, we present several recommendations to do so. Our protocol is applicable to analyses of all kinds of endogenous and exogenous RNAs, falling within length ranges of 20–30 and 50–70 nt, corresponding to microRNA and pre-microRNA lengths, respectively.
登录
查看更多内容
影响因子：
14.9
作者：
Starega-Roslan J;Krol J;Koscianska E;Kozlowski P;Szlachcic WJ;Sobczak K;Krzyzosiak WJ
通讯作者：
Krzyzosiak WJ
影响因子：
5.4
作者：
Cameron, Jennifer E.;Yin, Qinyan;Flemington, Erik K.
通讯作者：
Flemington, Erik K.
影响因子：
14.9
作者：
Válóczi, A;Hornyik, C;Havelda, Z
通讯作者：
Havelda, Z
DOI：
10.1504/ijbra.2009.028678
发表时间：
2009-01-01
影响因子：
--
作者：
Gomase, Virendra S;Parundekar, Akshay N
通讯作者：
Parundekar, Akshay N
影响因子：
4.8
作者：
Schmittgen, Thomas D.;Lee, Eun Joo;Chen, Caifu
通讯作者：
Chen, Caifu