利用荧光标记引物的 RT-PCR 进行拼接产物和异构体的高分辨率图谱绘制协议。

Protocol for High-Resolution Mapping of Splicing Products and Isoforms by RT-PCR Using Fluorescently Labeled Primers.

机构信息

Department of Chemistry and Biochemistry, University of California, Los Angeles, 607 Charles E. Young Drive East, Los Angeles, CA 90095-1569, USA.

出版信息

STAR Protoc. 2020 Oct 21;1(3):100140. doi: 10.1016/j.xpro.2020.100140. eCollection 2020 Dec 18.

DOI:10.1016/j.xpro.2020.100140

PMID:33377034

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7757285/

Abstract

We describe an RT-PCR protocol that allows high-resolution mapping of splicing products and isoforms using fluorescently labeled primers. Each species contains one fluorescent group allowing a direct comparison of the different isoforms despite size differences. A custom-size ladder enables the precise determination of cDNA lengths and discrimination of isoforms differing by less than five nucleotides on polyacrylamide gels. This protocol also allows the detection of products from splicing reactions, circumventing the need to use radiolabeled transcripts. For complete details on the use and execution of this protocol, please refer to Gabunilas and Chanfreau (2016).

摘要

我们描述了一种 RT-PCR 方案，该方案使用荧光标记引物允许对剪接产物和亚型进行高分辨率作图。每种物质包含一个荧光基团，允许直接比较不同的亚型，尽管存在大小差异。自定义大小的梯允许精确确定 cDNA 长度，并区分相差不到五个核苷酸的亚型在聚丙烯酰胺凝胶上。该方案还允许检测来自剪接反应的产物，从而避免使用放射性标记的转录本。有关此方案的使用和执行的完整详细信息，请参阅 Gabunilas 和 Chanfreau（2016 年）。