A protocol for extracting immunolabeled murine cardiomyocytes of high-quality RNA by laser capture microdissection.
A protocol for extracting immunolabeled murine cardiomyocytes of high-quality RNA by laser capture microdissection.
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DOI：
10.1016/j.xpro.2022.101231
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发表时间：
2022-03-18
期刊：
影响因子：
--
通讯作者：
Ellison-Hughes GM
中科院分区：
文献类型：
--
作者：
Shaalan AK;Ellison-Hughes GM
我们开发了一种高效的超短脉冲化学激光捕获显微切割（IHC-LMD）方案，该方案允许显微切割多达250个单个心肌细胞。在LMD之前，切除鼠心脏，快速冷冻并冷冻切片。从LMD材料中分离的RNA具有高RNA质量，使其可用于基因表达分析和RNA测序。该方案的挑战和限制包括PEN载玻片上的免疫染色和细胞核DAPI染料的可视化，以及尽可能限制RNA降解的时间和速度。LMD-从小鼠冷冻切片中获得免疫标记的心肌细胞适用于下一代测序分析的高质量RNA使用SYBR™绿色快速高级Cells-to-CT™技术进行基因表达分析我们开发了一种高效、超短的微化学-激光捕获显微切割（IHC-LMD）方案，该方案允许显微切割多达250个单个心肌细胞。在LMD之前，切除鼠心脏，快速冷冻并冷冻切片。从LMD材料中分离的RNA具有高RNA质量，使其可用于基因表达分析和RNA测序。该方案的挑战和限制包括PEN载玻片上的免疫染色和细胞核DAPI染料的可视化，以及尽可能限制RNA降解的时间和速度。
We developed a highly efficient, ultrashort immunohistochemistry-laser capture microdissection (IHC-LMD) protocol, which allows microdissection of up to 250 single cardiomyocytes. Before LMD, murine hearts are excised, snap-frozen, and cryosectioned. RNA isolated from LMD material is of high RNA quality, making it usable for gene expression analysis and RNA sequencing. Challenges and limitations of this protocol include visualization of the immunostaining and nuclei DAPI dye on the PEN slides, and timing and speed to limit RNA degradation as much as possible. LMD-procurement of immunolabeled cardiomyocytes from murine frozen sections High quality RNA suitable for next-generation sequencing analysis Gene expression analysis using SYBR™ Green Fast Advanced Cells-to-CT™ technology We developed a highly efficient, ultrashort immunohistochemistry-laser capture microdissection (IHC-LMD) protocol, which allows microdissection of up to 250 single cardiomyocytes. Before LMD, murine hearts are excised, snap-frozen, and cryosectioned. RNA isolated from LMD material is of high RNA quality, making it usable for gene expression analysis and RNA sequencing. Challenges and limitations of this protocol include visualization of the immunostaining and nuclei DAPI dye on the PEN slides, and timing and speed to limit RNA degradation as much as possible.
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