转录组分析]<em>活体</em>生产牛胚胎植入前胚胎采用双色微阵列平台
Summary
Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.
Abstract
Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.
Introduction
在高产奶牛胚胎早期损耗是在乳品行业1，2的主要挑战之一。牛已经成为一个有趣的模型来研究人类早期胚胎发育，由于其相似的发展过程3,4。然而，需要更多的研究来有关于涉及牛胚胎早期发育的基因更好的理解。
后由于第一微阵列技术二十年1995年5开发的，更为复杂的探测器制造工艺降低打印错误和内和不同微阵列平台6之间的阵列芯片可变性的发展。改进的微阵列技术导致了在临床研究7和广泛应用这种技术最近，在早期胚胎quality评估8。
大量的芯片技术所需的材料也就是为什么微阵列技术最初未能进入的一些研究领域，如早期胚胎发育的主要原因。最近，RNA扩增方法已得到改进，线性放大RNA为从起始原料RNA 9个子纳克微克的水平。目前市场上可用的几个商业RNA扩增试剂盒;然而，更多的受欢迎发达试剂盒相关的核糖单引物等温扩增10和T7启动子驱动的11种方法。最流行的反义RNA扩增的体外转录采用用寡dT引物在5'端12连结到一个T7启动子。该技术允许保持了最具代表性的反义转录物的线性扩增后˚F或阵列杂交13。该方法已适于放大来自牛胚胎8提取的总RNA皮克水平。
万向联动系统（ULS）是直接结合的DNA或扩增的RNA与铂联荧光色素或者花青547或花青647，通过形成于鸟嘌呤14的N7位置的配位键的标记方法。该方法适于在胚胎的研究，以产生更稳定的相比改性酶法15产生的aRNA的氨基烯丙基扩增的aRNA无需修改。单染料和两种染料标记方法已经在芯片使用通用联动系统调整。一个大型芯片的比较研究是有数据质量的一个和两个彩色阵列平台6之间的良好的相关性。
近来，T7启动子驱动Ñ反义RNA扩增和ULS标记方法已被开发，以提供更可靠的协议来产生标记的aRNA材料用于微阵列杂交8,16的高品质的足够量。因此，这项研究提供了一个协议来演示一些从RNA提取使用7天牛胚胎作为一个例子涉及双色微阵列数据分析的重要步骤。
Protocol
Representative Results
Discussion
第一个问题使用7天的牛胚胎是没有得到足够数量的高质量RNA是研究基因表达进行微阵列分析。不推荐用于第7天的胚胎传统苯酚/氯仿RNA提取和乙醇沉淀法，导致低产量以及可能剩苯酚抑制RNA扩增反应。相反，一个标准的基于列的方法是更好地隔离总RNA，然后洗脱以最小的洗脱缓冲液，以增加浓度的RNA。在目前的研究是利用相同的方法学26，目前所使用的相同的RNA提取试剂盒<sup cla…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Research supported by Alberta Livestock and Meat Agency, Alberta Innovates – BioSolutions, Alberta Milk, and Livestock Research Branch, Alberta Agriculture and Forestry.
Materials
| PicoPure RNA Isolation Kit | Applied Biosystems | KIT0204 | |
| RNase-Free DNase Set (50) | Qiagen | 79254 | |
| Agilent RNA 6000 Pico Kit | Agilent Technologies | 5067-1513 | |
| Arcturus RiboAmp HS PLUS Kit | Applied Biosystems | KIT0505 | |
| 2100 Bioanalyzer Instruments | Agilent Technologies | G2940CA | |
| RNA Screen Tape | Agilent Technologies | 5067-5576 | |
| ULS Fluorescent Labeling Kit | Kreatech Diagnostics | EA-021 | |
| Custom Gene Expression Microarrays | Agilent Technologies | G2514F | |
| Agilent Gene Expression wash buffer 1 | Agilent Technologies | Part #5188-5325 | |
| Agilent Gene Expression wash buffer 2 | Agilent Technologies | Part #5188-5326 | |
| 2X Hi-RPM Hybridization buffer | Agilent Technologies | Part #5190-0403 | |
| 25X Fragment buffer | Agilent Technologies | Part #5185-5974 | |
| 10X GE Blocking Agent | Agilent Technologies | Part #5188-5281 | |
| Stabilization and drying solution | Agilent Technologies | Part #5185-5979 | |
| Gasket slides enabled by Agilent SureHyb techonolgy | Agilent Technologies | G2524-60012 | Pack of 20 gasket slides, 4 microarrays/slide |
| Two-Color RNA Spike-In Kit | Agilent Technologies | Cat# 5188-5279 | |
| GenePix 4000B array scanner | Molecular Devices | GENEPIX 4000B-U | |
| Ozone Free Box | BioTray | OFB_100x200 | |
| GAL file | Agilent Technologies | – |
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