कस्टम MicroRNA माइक्रोएरे प्रयोगों प्रदर्शन
Summary
कस्टम microRNA माइक्रोएरे प्रयोगों प्रदर्शन करने का एक सरल प्रक्रिया में वर्णित है. चरणों में शामिल अलग शाही सेना, लेबलिंग शाही सेना और संदर्भ डीएनए, प्रोटीन के लिए नमूने hybridizing, प्रोटीन स्कैनिंग, बढ़ाता और संकरण संकेतों का विश्लेषण.
Abstract
microRNAs (miRNAs) are a large family of ˜ 22 nucleotides (nt) long RNA molecules that are widely expressed in eukaryotes 1. Complex genomes encode at least hundreds of miRNAs, which primarily inhibit the expression of a vast number of target genes post-transcriptionally 2, 3. miRNAs control a broad range of biological processes 1. In addition, altered miRNA expression has been associated with human diseases such as cancers, and miRNAs may serve as biomarkers for diseases and prognosis 4, 5. It is important, therefore, to understand the expression and functions of miRNAs under many different conditions.
Three major approaches have been employed to profile miRNA expression: real-time PCR, microarray, and deep sequencing. The technique of miRNA microarray has the advantage of being high-throughput, generally less expensive, and most of the experimental and analysis steps can be carried out in a molecular biology laboratory at most universities, medical schools and associated hospitals. Here, we describe a method for performing custom miRNA microarray experiments. A miRNA probe set will be printed on glass slides to produce miRNA microarrays. RNA is isolated using a method or reagent that preserves small RNA species, and then labeled with a fluorescence dye. As a control, reference DNA oligonucleotides corresponding to a subset of miRNAs are also labeled with a different fluorescence dye. The reference DNA will serve to demonstrate the quality of the slide and hybridization and will also be used for data normalization. The RNA and DNA are mixed and hybridized to a microarray slide containing probes for most of the miRNAs in the database. After washing, the slide is scanned to obtain images, and intensities of the individual spots quantified. These raw signals will be further processed and analyzed as the expression data of the corresponding miRNAs. Microarray slides can be stripped and regenerated to reduce the cost of microarrays and to enhance the consistency of microarray experiments. The same principles and procedures are applicable to other types of custom microarray experiments.
Protocol
Discussion
गहरी अनुक्रमण प्रौद्योगिकियों में हाल के अग्रिमों के बावजूद, माइक्रोएरे डीएनए और आरएनए के उच्च throughput विश्लेषण के लिए एक व्यवहार्य विकल्प रहता है. गहरी अनुक्रमण की तुलना में, माइक्रोएरे प्रयोगों सस्ता …
Disclosures
The authors have nothing to disclose.
Acknowledgements
काम के हिस्से में नशीली दवाओं के सेवन केंद्र के राष्ट्रीय संस्थान (P50 011806 डीए) और संयुक्त राज्य अमेरिका सेना के रक्षा विभाग (W81XWH-07-1-0183) द्वारा समर्थित किया गया.
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 |
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