Protocol for High-Resolution Mapping of Splicing Products and Isoforms by RT-PCR Using Fluorescently Labeled Primers.
Protocol for High-Resolution Mapping of Splicing Products and Isoforms by RT-PCR Using Fluorescently Labeled Primers.
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DOI：
10.1016/j.xpro.2020.100140
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发表时间：
2020-12-18
期刊：
影响因子：
--
通讯作者：
Chanfreau GF
中科院分区：
文献类型：
--
作者：
Weathers I;Gabunilas J;Samson J;Roy K;Chanfreau GF
我们描述了一个RT-PCR协议，允许高分辨率映射的剪接产物和异构体使用荧光标记的引物。每个物种包含一个荧光基团，允许直接比较不同的亚型，尽管大小不同。自定义大小的阶梯能够精确测定cDNA长度，并在聚丙烯酰胺凝胶上区分差异小于5个核苷酸的同种型。该方案还允许检测来自体外剪接反应的产物，从而避免了使用放射性标记的转录物的需要。有关本方案使用和执行的完整详细信息，请参阅。以高分辨率检测剪接和未剪接异构体的RT-PCR方案使用Cy 3标记的引物避免了放射性的使用包括制备Cy 3标记的定制大小梯状蛋白的详细说明方案可用于检测体内和体外剪接产物我们描述了一种RT-PCR方案，该方案允许使用荧光标记的引物对剪接产物和异构体进行高分辨率作图。每个物种包含一个荧光基团，允许直接比较不同的亚型，尽管大小不同。自定义大小的阶梯能够精确测定cDNA长度，并在聚丙烯酰胺凝胶上区分差异小于5个核苷酸的同种型。该方案还允许检测来自体外剪接反应的产物，从而避免了使用放射性标记的转录物的需要。
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 in vitro splicing reactions, circumventing the need to use radiolabeled transcripts. For complete details on the use and execution of this protocol, please refer to. RT-PCR protocol to detect spliced and unspliced isoforms at high resolution Use of Cy3-labeled primers circumvents the use of radioactivity Includes detailed instructions to prepare a Cy3-labeled custom-size ladder Protocol can be applied to detect spliced products in vivo and in vitro 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 in vitro splicing reactions, circumventing the need to use radiolabeled transcripts.
影响因子：
4.5
作者：
Gabunilas J;Chanfreau G
通讯作者：
Chanfreau G
影响因子：
4.5
作者：
Kawashima T;Douglass S;Gabunilas J;Pellegrini M;Chanfreau GF
通讯作者：
Chanfreau GF
影响因子：
4.5
作者：
Kawashima, Tadashi;Pellegrini, Matteo;Chanfreau, Guillaume F.
通讯作者：
Chanfreau, Guillaume F.