对基因差异表达的RNA序列中电穿孔鸡胚脊髓分析
Summary
This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.
Abstract
In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.
Introduction
在活的生物体的基因研究经常使用的鸡胚为模型，因为在OVO电代表了一个快速和廉价的方式来部分地转染胚胎结构在体内与DNA构建1-5。编码含有荧光报告与感兴趣的基因，如pCIG 6或机动设备7在一起的一个双顺反子转录物表达载体，允许在所希望的区域转质量的快速验证处理的胚胎用于下游分析之前，在立体显微镜下。
在DNA测序的最新进展，现在有可能从使用RNA测序-8,9- RNA样本获得数字全转录组表达图谱。因此，代替费时方法，使只有几个基因产物平行，分析如原位杂交，免疫组织化学和qPCR，用于制备cDNA文库的高通量测序可以提供全转录组的信息。对每一个基因的表达水平的实验效果的观察又可以提供关于所使用的构建体改变了通路有力的见解。这是特别容易的，如果一个基因组装配所使用的生物体是可用的，从而鸡胚又是一个合适的模型。
如果用户有访问权限的可靠的基因组测序中心，主要问题是获得高品质的RNA样品。这项工作表明获得的RNA样品从转染的神经管适用于RNA测序，以及在下游步骤中所得到的数据集的计算机分析的方法。电在HH12-13其次是48小时培养后，转染树干脊髓半解剖的核糖核酸酶自由的条件下，立即溶解并进一步降解保护的超低冰点，直到RNA纯化和图书馆preparat离子。
银河公共服务器10可以访问免费资源，用于分析的高通量测序数据。的空间供注册用户的数量是足够的小实验，从而省去了本地的计算基础设施。 RNA测序数据分析包括过滤，比对和基因表达的定量/对比。虽然有用于RNA测序数据分析的一般准则，滤波参数将在很大程度上取决于序列的质量，并应后的原始数据的质量控制分析来确定。
这个协议描述的步骤，以获得与从鸡胚胎转染体内脊髓比较RNA片段轮廓。作为代表性的结果，从转染了空载体两个独立的对照样品获得的数据集的比较表明，该方法是可重复的，应该允许鉴定差异EXPRES的SED成绩单实验样本。因此，该方法的应用有可能大大增加的发现的数量之后，一个单独的试验，从而提供一个大的数据集进行后续调查。
Protocol
Representative Results
Discussion
在这里，我们为鸡脊髓电后分析效果的准则。虽然DNA载体进行电穿孔是更频繁地用于过表达感兴趣的基因，还可以使用构建体编码显性阴性，quimeric蛋白或前体的siRNA，以产生基因功能敲低的条件19-21。事实上，从两个GAIN-和丧失功能的分析得到的转录概况比较可以指出差异表达的基因在相反的方式中的每个条件，因此具有潜在地发现更多的具体效果。
如任何方法下游?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
CYIY is supported by FAPESP (2012/14421-5) and FMV is supported by a fellowship from FAPESP (2009/53695-0). We thank the DNA Technologies Core at University of California, Davis for preparation and sequencing of the RNA-Seq libraries used in this work, the Galaxy team for providing an excellent and free interface for high-throughput sequencing data analysis tools and Dr. Marianne Bronner for allowing us to film in her laboratory space.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Indian ìnk | Any supplier | ||
| 18G x 1 1/2 needle | BD | 305196 | |
| 3 ml syringe | BD | 309657 | |
| Tris | MP Biomedicals | 819620 | |
| F D & C Blue 1 | Spectra Colors Corporation | 5.FC.0010P0 | Food Dye used to visualize DNA injection |
| Ringer's solution (1 L) | |||
| – 7.2 g NaCl | Sigma-Aldrich | S-9888 | |
| – 0.37 g KCl | Sigma-Aldrich | P-4504 | |
| – 0.225 g CaCl2.2H2O | Fisher Scientific | 10043-52-4 | |
| – 0.217 g Na2HPO4.7H2O | Sigma-Aldrich | S-9390 | |
| – 0.02 g KH2PO4 | Sigma-Aldrich | P-5379 | |
| – ddH2O to 800 ml and adjust pH to 7.4 | |||
| – ddH2O to 1 L | |||
| – Filter and autoclave | |||
| PBS (Phosphate Buffered Solution) (1 L) | |||
| – 8 g NaCl | Sigma-Aldrich | S-9888 | |
| – 0.2 g KCl | Sigma-Aldrich | P-4504 | |
| – 1.15 g Na2HPO4.7H2O | Sigma-Aldrich | S-9390 | |
| – 0.2 g KH2PO4 | Sigma-Aldrich | P-5379 | |
| – ddH2O to 800 ml and adjust pH to 7.2 | |||
| – ddH2O to 1 L and filter | |||
| – 1 ml DEPC (Diethylpyrocarbonate) | Sigma-Aldrich | D-5758 | |
| – Shake vigorously and let stand overnight at room temperature | |||
| – Autoclave | |||
| Sieved spoon | Any supplier | ||
| Sterile 60 x 15 mm polystirene Petri dishes | Corning Life Sciences | 351007 | |
| RNaseZap RNase decontamination solution | Life Technologies | AM9780 | |
| Fine point surgical scissors | Any supplier | ||
| Straight fine point tweezers | Any supplier | ||
| Pulled glass needle made from 1.1 x 75 mm glass capillary tubes | Kimble Chase | 40A502 | |
| 9'' glass Pasteur pipette | Any supplier | ||
| Manual pipette pump | Any supplier | ||
| Clear 1.5 ml microcentrifuge polypropilene tubes | Corning Life Sciences | MCT-150-C | |
| Microcentrifuge | Any supplier | ||
| RNAlater solution for RNA stabilization | Life Technologies | AM7020 | |
| RNAqueous total RNA isolation kit | Life Technologies | AM1912 | |
| Molecular Biology Grade Ethanol | Any supplier | ||
| RNA 6000 Nano Kit | Agilent Technologies | 5067-1511 | |
| 2100 Electrophoresis Bioanalyzer Instrument | Agilent Technologies | G2939AA | |
| Truseq RNA Sample Preparation Kit v2 | Illumina | RS-122-2001/RS-122-2002 |
References
- Itasaki, N., Bel-Vialar, S., Krumlauf, R. Shocking’ developments in chick embryology: electroporation and in ovo gene expression. Nat. Cell Biol. 1 (8), 207-20 (1999).
- Krull, C. E. A primer on using in ovo electroporation to analyze gene function. Dev. Dyn. 229 (3), 433-439 (2004).
- Blank, M. C., Chizhikov, V., Millen, K. J. In ovo electroporations of HH stage 10 chicken embryos. J. Vis. Exp. (9), 10-3791 (2007).
- Sauka-Spengler, T., Barembaum, M. Gain- and loss-of-function approaches in the chick embryo. Methods Cell Biol. 87, 237-256 (2008).
- Ghadge, G. D., et al. Truncated wild-type SOD1 and FALS-linked mutant SOD1 cause neural cell death in the chick embryo spinal cord. Neurobiol. Dis. 21 (1), 205-20 (2006).
- Megason, S. G., McMahon, A. P. A mitogen gradient of dorsal midline Wnts organizes growth in the CNS. Development. 129 (9), 2087-2098 .
- Swartz, M., Eberhart, J., Mastick, G. S., Krull, C. E. Sparking new frontiers: using in vivo electroporation for genetic manipulations. Dev. Biol. 233 (1), 13-21 (2001).
- Cloonan, N., et al. Stem cell transcriptome profiling via massive-scale mRNA sequencing. Nat. Methods. 5 (7), 613-619 (2008).
- Mortazavi, A., Williams, B. A., McCue, K., Schaeffer, L., Wold, B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat. Methods. 5 (7), 621-628 (2008).
- Goecks, J., Nekrutenko, A., Taylor, J. Galaxy Team. Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences. Genome Biol. 11 (8), 86 (2010).
- Trichas, G., Begbie, J., Srinivas, S. Use of the viral 2A peptide for bicistronic expression in transgenic mice. BMC Biol. 6, 40 (2008).
- Maniatis, T., Fritsch, E. F., Sambrook, J. . Molecular cloning. A laboratory manual. , (1982).
- Wang, Y., et al. Evaluation of the coverage and depth of transcriptome by RNA-Seq in chickens. BMC Bioinformatics. 12, 10-1186 (2011).
- Blankenberg, D., et al. Manipulation of FASTQ data with Galaxy. Bioinformatics. 26 (14), 1783-1785 (2010).
- Kim, D., Pertea, G., Trapnell, C., Pimentel, H., Kelley, R., Salzberg, S. L. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14 (4), 36 (2013).
- Trapnell, C., et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol. 28 (5), 511-515 (2010).
- Quinlan, A. R., Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 26 (6), 841-842 (2010).
- Vieceli, F. M., Simões-Costa, M., Turri, J. A., Kanno, T., Bronner, M., Yan, C. Y. The transcription factor chicken Scratch2 is expressed in a subset of early postmitotic neural progenitors. Gene Expr. Patterns. 13 (5-6), 189-196 (2013).
- Chesnutt, C., Niswander, L. Plasmid-based short-hairpin RNA interference in the chicken embryo. Genesis. 39 (2), 73-78 (2004).
- Katahira, T., Nakamura, H. Gene silencing in chick embryos with a vector-based small interfering RNA system. Dev. Growth. Differ. 45 (4), 361-367 (2003).
- Rao, M., Baraban, J. H., Rajaii, F., Sockanathan, S. In vivo comparative study of RNAi methodologies by in ovo electroporation in the chick embryo. Dev. Dyn. 231 (3), 592-600 (2004).
- Lu, Y., Lin, C., Wang, X. PiggyBac transgenic strategies in the developing chicken spinal cord. Nucleic Acids Res. 37 (21), 141 (2009).
- Simões-Costa, M., Tan-Cabugao, J., Antoshechkin, I., Sauka-Spengler, T., Bronner, M. E. Transcriptome analysis reveals novel players in the cranial neural crest gene regulatory network. Genome Res. 24 (2), 281-290 (2014).
- Trapnell, C., et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7 (3), 562-578 (2012).
