执行自定义的微RNA基因芯片实验
Summary
一个简单的程序执行自定义的microRNA微阵列实验描述。这些步骤包括隔离RNA，标签的RNA和参照DNA样本，杂交到芯片，芯片扫描，量化和分析杂交信号。
Abstract
小分子RNA（miRNAs）是一个大家族〜22核苷酸（nt）长的RNA分子，在真核生物中 1广泛表达。复杂的基因组编码至少有数百个miRNA的，这主要是抑制大量的靶基因的转录后2，3的表达。 miRNA的控制范围广泛的生物过程1。此外，miRNA表达的改变已经与人类疾病，如癌症，和miRNA可作为生物标志物疾病和预后4， 5。因此，重要的是理解表达和许多不同的条件下miRNA的功能。
已采用文件miRNA表达的实时PCR技术，基因芯片和深度测序的三个主要方法。的miRNA芯片技术具有高通量的优势，一般较便宜，和大多数的实验和分析的步骤可卡尔IED在分子生物学实验室，在大多数大学，医学院校和相关的医院。在这里，我们描述了一个用于执行自定义的miRNA微阵列实验的方法。一个miRNA探针组将印上产生的miRNA微阵列玻片。 RNA是隔离的使用方法或试剂，保留小分子RNA的物种，然后用荧光染料标记。作为对照，参考相应的DNA寡核苷酸的miRNA的一个子集上，也有不同的荧光染料。参照DNA将成为展示的幻灯片和杂交质量，也将用于数据规范化。 RNA和DNA混合并杂交到芯片的幻灯片，其中包含数据库中的大多数miRNA的探针。洗涤后，幻灯片扫描获取图像，个别点强度量化。这些原始信号将得到进一步的处理和相应的miRNA表达数据分析。微安rray幻灯片可以剥离和再生，以降低芯片的成本和提高的微阵列实验的一致性。相同的原则和程序适用于其他类型的定制微阵列实验。
Protocol
Discussion
尽管深度测序技术的最新进展，芯片仍然是一个可行的选择，为高通量DNA和RNA分析。深度测序相比，微阵列实验比较便宜，一个典型的分子生物学实验室可以执行大部分的实验和数据分析的房子，允许灵活性和节省时间。在未来的芯片可能非常适合集中审问的基因，例如集，全部或在基因组或miRNA的转录因子的一个子集，这里提出相同的原则和程序可以用于定制微阵列实验研究此外miRNA的基因家族…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国立药物滥用中心（011806 P50的DA）和美国军队国防部（W81XWH – 07 – 1 – 0183）中的一部分。
Materials
| Items | Vendor | Catalogue number | Comments |
|---|---|---|---|
| NCode Multi-Species miRNA Microarray Probe Set V2 | Invitrogen | MIRMPS201 | Designed based on the miRBase Release 9.0 (October 2006). It contains ˜ 1,140 unmodified, 34-44 nt long oligonucleotides as probes for worm, fly, zebrafish, mouse, rat, and human miRNAs, and a number of internal control probes such as snoRNAs. The miRNA probes are doublets of the sequences complementary to mature miRNAs, hence the size of ˜ 44 nt. For analysis one can focus on miRNAs from a particular genome(s) of interest. |
| Trizol | Invitrogen | 15596018 | We have also used enriched, small RNA fraction for labeling, although total RNA samples are faster and easier to prepare and to quantify and suitable for downstream applications such as mRNA analysis. |
| T4 RNA Ligase 1 | New England Biolabs | M0204L | |
| Ulysis Alexa Fluor 647 Nucleic Acid Labeling Kit | Invitrogen | U21660 | This kit or similar products can be used to label experimental RNA samples or a control RNA (instead of control DNA) as well. |
| 5’-pCU-DY547-3’ | Dharmacon | Custom made | Small RNA fraction can be similarly labeled by ligation. |
| CentriSep columns | Princeton Separations | CS-901 | |
| GAPSII coated slide | Corning | 40004 | Other types of slides may be also used. |
| Microarray hybridization chambers | Corning | 2551 or 40080 | Other kinds of hybridization chambers and coverslips should also work. Using commercially available hybridization machines can reduce hybridization time significantly, e.g., to ˜ 2 hours. |
| Lifterslips | Thermo/Erie Scientific | 25X60I-M5439-001-LS | |
| BlueFuse | BlueGenome | ||
| GeneSpring | Agilent |
References
- Ambros, V. The functions of animal microRNAs. Nature. 431, 350-355 (2004).
- Friedman, R. C., Farh, K. K., Burge, C. B., Bartel, D. P. Most mammalian mRNAs are conserved targets of microRNAs. Genome Res. 19, 92-105 (2009).
- Chekulaeva, M., Filipowicz, W. Mechanisms of miRNA-mediated post-transcriptional regulation in animal cells. Curr. Opin. Cell Biol. 21, 452-460 (2009).
- Farazi, T. A., Spitzer, J. I., Morozov, P., Tuschl, T. miRNAs in human cancer. J. Pathol. 223, 102-115 (2011).
- Small, E. M., Olson, E. N. Pervasive roles of microRNAs in cardiovascular biology. Nature. 469, 336-342 (2011).
- Thomson, J. M., Parker, J., Perou, C. M., Hammond, S. M. A custom microarray platform for analysis of microRNA gene expression. Nat. Methods. 1, 47-53 (2004).
- Zhang, X., Xu, W., Tan, J., Zeng, Y. Stripping custom microRNA microarrays and the lessons learned about probe:slide interactions. Anal. Biochem. 386, 222-227 (2009).
- Griffiths-Jones, S., Saini, H. K., van Dongen, S., Enright, A. J. miRBase: tools for microRNA genomics. Nucl. Acids. Res. 36, D154-D158 (2008).
- Landgraf, P. A mammalian microRNA expression atlas based on small RNA library sequencing. Cell. 129, 1401-1414 (2007).
- Chiang, H. R. Mammalian microRNAs: experimental evaluation of novel and previously annotated genes. Genes. Dev. 24, 992-1009 (2010).
