Name: performing custom microrna microarray experiments
Uploaded: 2011-10-28T12:00:00
Description: Watch this Scientific Journal Video about Performing Custom MicroRNA Microarray Experiments at JoVE.com

The work was supported in part by National Institute of Drug Abuse Center (P50 DA 011806) and United States Army Department of Defense (W81XWH-07-1-0183).

1. Printing of custom miRNA microarrays
<ol>
	<li>Print the microarray slides using the microarray core facility services at a university or a company. The quality of microarray fabrication is one of the most critical factors for the success of a microarray experiment. Try a few services if possible. Ideally, one would print 50-100 slides at a time, and all the slides will look identical, with well separated, individual spots.</li>
	<li>For microarray support, we use the GAPS II coated slides.</li>
	<li>For miRNA probe...

<ol>
	<li>Ambros, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functions+of+animal+microRNAs.">The functions of animal microRNAs.</a> Nature. 431, 350-355 (2004).</li><li>Friedman, R. C., Farh, K. K., Burge, C. B., Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Most+mammalian+mRNAs+are+conserved+targets+of+microRNAs.">Most mammalian mRNAs are conserved targets of microRNAs.</a> Genome Res. 19, 92-105 (2009).</li><li>Chekulaeva, M., Filipowicz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+miRNA-mediated+post-transcriptional+regulation+in+animal+cells.">Mechanisms of miRNA-mediated post-transcriptional regulation in animal cells.</a> Curr. Opin. Cell Biol. 21, 452-460 (2009).</li><li>Farazi, T. A., Spitzer, J. I., Morozov, P., Tuschl, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRNAs+in+human+cancer.">miRNAs in human cancer.</a> J. Pathol. 223, 102-115 (2011).</li><li>Small, E. M., Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pervasive+roles+of+microRNAs+in+cardiovascular+biology.">Pervasive roles of microRNAs in cardiovascular biology.</a> Nature. 469, 336-342 (2011).</li><li>Thomson, J. M., Parker, J., Perou, C. M., Hammond, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+custom+microarray+platform+for+analysis+of+microRNA+gene+expression.">A custom microarray platform for analysis of microRNA gene expression.</a> Nat. Methods. 1, 47-53 (2004).</li><li>Zhang, X., Xu, W., Tan, J., Zeng, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stripping+custom+microRNA+microarrays+and+the+lessons+learned+about+probe:slide+interactions.">Stripping custom microRNA microarrays and the lessons learned about probe:slide interactions.</a> Anal. Biochem. 386, 222-227 (2009).</li><li>Griffiths-Jones, S., Saini, H. K., van Dongen, S., Enright, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRBase:+tools+for+microRNA+genomics.">miRBase: tools for microRNA genomics.</a> Nucl. Acids. Res. 36, D154-D158 (2008).</li><li>Landgraf, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mammalian+microRNA+expression+atlas+based+on+small+RNA+library+sequencing.">A mammalian microRNA expression atlas based on small RNA library sequencing.</a> Cell. 129, 1401-1414 (2007).</li><li>Chiang, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+microRNAs:+experimental+evaluation+of+novel+and+previously+annotated+genes.">Mammalian microRNAs: experimental evaluation of novel and previously annotated genes.</a> Genes. Dev. 24, 992-1009 (2010).</li></ol>

Despite recent advances in deep sequencing technologies, microarray remains a viable choice for high-throughput analysis of DNA and RNA. Compared to deep sequencing, microarray experiments are cheaper, and a typical molecular biology laboratory can perform most of the experiments and data analysis in-house, which allows for flexibility and saves time. In the future, microarrays are likely well-suited to intensively interrogate sets of genes, e.g., all or a subset of the transcription factors in a genome or miRNAs, and ...

<table border="1">
<tbody>
<tr>
<th>Items</th>
<th>Vendor</th>
<th>Catalogue number</th>
<th>Comments</th>
</tr>
<tr>
<td>NCode Multi-Species miRNA Microarray Probe Set V2</td>
<td>Invitrogen</td>
<td>MIRMPS201</td>
<td>Designed based on the miRBase Release 9.0 (October 2006). It contains &tilde; 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 &tilde; 44 nt. For analysis one can focus on miRNAs from a particular genome(s) of interest.</td>
</tr>
<tr>
<td>Trizol </td>
<td>Invitrogen</td>
<td>15596018</td>
<td>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.</td>
</tr>
<tr>
<td>T4 RNA Ligase 1</td>
<td>New England Biolabs</td>
<td>M0204L</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Ulysis Alexa Fluor 647 Nucleic Acid Labeling Kit</td>
<td>Invitrogen</td>
<td>U21660</td>
<td>This kit or similar products can be used to label experimental RNA samples or a control RNA (instead of control DNA) as well.</td>
</tr>
<tr>
<td>5&#x2019;-pCU-DY547-3&#x2019;</td>
<td>Dharmacon</td>
<td>Custom made</td>
<td>Small RNA fraction can be similarly labeled by ligation. </td>
</tr>
<tr>
<td>CentriSep columns </td>
<td>Princeton Separations </td>
<td>CS-901</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>GAPSII coated slide</td>
<td>Corning</td>
<td>40004</td>
<td>Other types of slides may be also used.</td>
</tr>
<tr>
<td>Microarray hybridization chambers</td>
<td>Corning</td>
<td>2551 or 40080</td>
<td>Other kinds of hybridization chambers and coverslips should also work. Using commercially available hybridization machines can reduce hybridization time significantly, e.g., to &tilde; 2 hours. </td>
</tr>
<tr>
<td>Lifterslips</td>
<td>Thermo/Erie Scientific</td>
<td>25X60I-M5439-001-LS</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>BlueFuse</td>
<td>BlueGenome</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>GeneSpring</td>
<td>Agilent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
</tbody>
</table>

performing custom microrna microarray experiments

microRNAs (miRNAs) are a large family of &tilde; 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.

Watch this Scientific Journal Video about Performing Custom MicroRNA Microarray Experiments at JoVE.com

Performing Custom MicroRNA Microarray Experiments

A simple procedure of performing custom microRNA microarray experiments is described. The steps include isolating RNA, labeling RNA and reference DNA, hybridizing the samples to microarrays, scanning the microarrays, quantifying and analyzing hybridization signals.

microRNAs (miRNAs) are a large family of &tilde; 22 nucleotides (nt) long RNA molecules that are widely expressed in eukaryotes 1.  Complex genomes encode ...

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