A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones
A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones
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DOI：
10.3791/60197
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发表时间：
2019-09-01
影响因子：
1.2
通讯作者：
Erben, Reinhold G.
中科院分区：
文献类型：
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作者：
Marek, Ana;Schueler, Christiane;Erben, Reinhold G.
RNA产量和完整性对于RNA分析是决定性的。然而，在整个激光捕获显微切割（LCM）过程中保持RNA的完整性通常在技术上具有挑战性。由于LCM研究使用少量材料，因此对有限RNA产量的关注也很重要。因此，开发了LCM方案以获得足够量的高质量RNA用于骨细胞中的基因表达分析。评价了染色方案、冷冻切片厚度、显微切割组织数量、RNA提取试剂盒和LCM系统对从显微切割骨细胞获得的RNA产量和完整性的影响。使用粘合膜制作8 μ m厚的冷冻骨切片，并使用商业LCM染色的快速方案进行染色。将样品夹在聚对苯二甲酸乙二醇酯（PET）膜和粘合剂膜之间。使用重力进行样品收集的LCM系统和基于柱的RNA提取方法来获得足够产量的高质量RNA。目前的研究重点是小鼠股骨切片。然而，这里报道的LCM方案可用于研究在生理条件和疾病过程中任何硬组织细胞中的原位基因表达。
RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser capture microdissection (LCM) procedure. Since LCM studies work with low amounts of material, concerns about limited RNA yields are also important. Therefore, an LCM protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The effect of staining protocol, thickness of cryosections, microdissected tissue quantity, RNA extraction kit, and LCM system used on RNA yield and integrity obtained from microdissected bone cells was evaluated. Eight-mu m-thick frozen bone sections were made using an adhesive film and stained using a rapid protocol for a commercial LCM stain. The sample was sandwiched between a polyethylene terephthalate (PET) membrane and the adhesive film. An LCM system that uses gravity for sample collection and a column-based RNA extraction method were used to obtain high quality RNAs of sufficient yield. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study in situ gene expression in cells of any hard tissue in both physiological conditions and disease processes.