大规模的斑马鱼胚胎的心脏解剖的转录分析
Summary
到斑马鱼心脏发育过程中分析的心脏基因表达分布，总RNA已被提取从离体心脏。在这里，我们提出了一个协议，用于收集功能/跳动的心迅速人工清扫，从斑马鱼胚胎获得心脏特异性mRNA。
Abstract
斑马鱼胚胎的心脏是由只有几百细胞，占整个胚胎中只有一小部分。因此，为了防止被掩蔽由全局胚胎转录心脏转录，有必要收集足够数量的心作进一步的分析。此外，如斑马鱼心脏发育迅速进行，心脏收集和RNA提取方法需要快，以保证样品的均匀性。在这里，我们提出了一个快速的手动清扫协议收集功能/跳动的心，从斑马鱼胚胎。这是为随后的心脏特异性RNA提取，以确定心肌特异性基因表达水平通过转录组分析，例如定量实时聚合酶链反应（RT-qPCR的）的必要先决条件。该方法是基于在斑马鱼胚胎心脏和其他组织相比的差的粘合性能;这允许迅速的phy从心外心脏组织由流体剪切力的破坏，逐步过滤和人工采集转基因荧光标记心中的组合SICAL分离。
Introduction
斑马鱼（ 斑马鱼 ）被广泛用于发育生物学研究器官体内 ，由于其快速的，透明的和宫外胚胎发育，结合小尺寸和转基因记者线荧光蛋白的组织特异性表达的可用性。这个小脊椎动物特别适于研究心脏发育，因为早期的斑马鱼胚胎的氧合不依赖于心脏的跳动及血液流动;这些特点使大量心血管突变1,2的表征和斑马鱼现在是一个被广泛认可的模式生物来研究心脏疾病3。
到胚胎发育过程中研究基因表达，转录通常通过整个安装原位杂交（WISH）4分析，RT-qPCR的5，微阵列6，或下一代测序（RNA测序）7。而WISH允许基因表达的整个胚胎内的空间 – 时间分析，转录水平通过RT-qPCR的，微阵列或RNA测序方法，通常进行评估。然而，这些方法需要组织富集为特定的基因表达图谱。
因为斑马鱼胚胎心脏表示整个胚胎的一小部分，心脏发育过程中转录研究需要一个协议，用于解剖和心脏的富集。此外，为了得到生理上相关的数据，以维持心脏组织完全功能，直到RNA提取它是很重要的。在这里，我们描述了一种协议，用于快速分离生理正常和跳动心脏从数百斑马鱼胚胎的有效地获得高品质的RNA样品用于进一步分析。本方法是基于报道的烧伤和麦克雷，2006年8所述的协议，对于心脏组织的富集，这两种方法都使用的转基因心肌记者线并利用斑马鱼胚胎心脏与其他组织的鉴别粘附性能优势。简要地说，通过一个狭窄的枪头吹打许多胚胎上下，心脏被同时从胚体释放并随后从胚胎碎片两个快速过滤步骤中分离;荧光标记的心脏，然后手动地从剩余的碎片来分类并收集用于进一步处理。
Protocol
Representative Results
Discussion
该协议允许斑马鱼胚胎心脏组织的快速富集的基因表达分析。心脏特异性RNA样品的数量和质量在很大程度上取决于几个关键步骤：第一，如果能够防止在该协议中的每一步的心损耗的样品的量有很大的提高，因为RNA纯化将仅具有足够的工作起始材料。第二，样品，这完全取决于实验者的纯度，通过分类和收集的心内的培养皿同时严格排除其它组织确定的。第三，样品的质量主要取决于限制可能发?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We would like to thank C. Burns, C. McRae for outlining the basic principle of this purification protocol, and F. Priller for initial implementation of this method in our lab. S.A.-S. is supported by a Heisenberg professorship of the Deutsche Forschungsgemeinschaft (DFG). This work was supported by DFG grant SE2016/7-1.
Materials
| Equipment for raising fish and collecting eggs | see the Zebrafish Book11 for details | ||
| Fluorescence stereomicroscope | |||
| Refrigerated Microcentrifuge | |||
| UV-Spectrophotometer | eg. Thermo Scientific Nanodrop 2000 | ||
| Nucleic acid electrophoresis chamber | |||
| Petri dishes 4cm Ø, coated with 1% agarose in E3 medium | |||
| Micropipettes and tips (P20, P100, P1000) | |||
| 1,5ml centrifugation tubes | |||
| 15ml and 50ml centrifugation tubes | |||
| Pair of Dumont #5 forceps | |||
| ExactaCruz™ Round Gel Loading Tips in Sterile Rack, 1-200μl | Santa Cruz | sc-201732 | |
| 100 μm filter (BD Falcon 100 mm Cell Strainer) | BD Biosciences | 352360 | |
| 30 μm filter (Pre-Separation Filters-30 µm) | Miltenyi Biotec | 130-041-407 | |
| Phase lock gel, heavy, 1,5ml tubes | Prime | 2302810 | |
| Egg water medium | 60μg/ml Instant Ocean Sea Salts in ddH2O, 0.00001% (w/v) Methylene Blue | ||
| E3 medium | 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4 | ||
| Tricaine (3-amino benzoic acidethylester) | Sigma-Aldrich | A-5040 | 4mg/ml Tricaine stock solution, pH 7 |
| 1% agarose (in E3 medium) | |||
| 1% agarose gel (in TBE buffer) | |||
| Leibovitz´s L-15 medium | Gibco | 21083-027 | |
| FBS (Fetal Bovine Serum) | Sigma | F4135 | |
| RNAlater | Ambion | AM7020 | |
| Trizol | Ambion | 1559606 | |
| Glycogen | Invitrogen | 10814-010 | 20 µg/µL in RNase-free water |
| chloroform | |||
| isopropanol | |||
| 75% ethanol (in DEPC-ddH2O) | |||
| Nuclease-free water or sterilized DEPC treated ddH2O | |||
| Nucleic acid loading buffer | |||
| TBE (Tris/Borate/EDTA) buffer for electrophoresis |
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