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 probes, we use the NCode Multi-Species miRNA Microarray Probe Set V2.</li>
	<li>Dissolve all the oligonucleotides in 3 x SSC at 10 &mu;M and quadruply print them on the slides. Fix the slides by ultraviolet irradiation according to instructions for the GAAPS II slides. Label the slide with a diamond pen to mark the area and side with the printed probes. Store at 22&deg;C.</li>
</ol>
2. Sample preparation
<ol>
	<li>Any method or reagent can be used to isolate RNA, as long as it preserves small RNAs. We generally use Trizol (Invitrogen) to extract total RNA, with satisfactory quantity and quality of RNA preparations, as measured by A260nm and A280nm readings.</li>
	<li>For RNA labeling, mix &tilde; 25 &mu;g of total RNA with 0.5 &mu;g of 5'-pCU-DY547-3' 6 in 1 x buffer (50 mM HEPES, pH 7.8, 20 mM MgCl2, 10 &mu;g/ml BSA, 10% DMSO) containing &tilde; 20 units T4 RNA Ligase 1, supplemented with &tilde; 10 mM DTT and &tilde; 0.02-0.03 final volume of the 10 x T4 RNA Ligase 1 buffer for ATP.</li>
	<li>Allow labeling to proceed on ice inside a refrigerator for 2-24 hours. Precipitate RNA with 0.3 M sodium acetate and 3 volumes of ethanol. For efficient ligation and precipitation, dissolve total RNA at over 2 mg/ml, and keep the total ligation volume at &tilde; 20 &mu;l.
	<ol>
		<li>If less RNA is available, the amount of input RNA and 5'-pCU-DY547-3' can be scaled down.</li>
		<li>If the RNA is very dilute, add carrier such as yeast tRNA to aid in precipitation.</li>
	</ol>
	</li>
	<li>Combine and label in total 1 &mu;g of reference DNA oligonucleotides corresponding to a subset of mammalian miRNAs using the Ulysis Alexa Fluor 647 Nucleic Acid Labeling Kit 6 as a control for the hybridization process. Purify the labeled DNA using a CentriSep column and save in dark at -20&deg;C.</li>
</ol>
3. Microarray hybridization
<ol>
	<li>To use the slides for the first time, pre-hybridize the slides in a filtered solution of 3 x SSC, 0.1% SDS, 0.2% BSA for 30-60 min at 37 &deg;C. Submerge them in water a few times, then in isopropanol. Dry the slides using a centrifuge with a slide adapter at 100 g for 5 min at 22&deg;C.</li>
</ol>
Note: We use Corning microarray hybridization chambers and Erie Scientific's mSeries of Lifterslips to perform microarray hybridization.
<ol start="2">
	<li>Rinse the lifterslips in water, then in ethanol, before drying in air. Place a slide inside a microarray hybridization chamber base, and a lifterslip on top of the slide. The lifterslip will cover the probe area, with its white strips contacting the slide.</li>
	<li>In the dark, spin down the precipitated, labeled RNA. Wash it once with 70% ethanol, and air dry the pellet. The pellet should be reddish.</li>
	<li>Prepare a hybridization solution containing 400 mM Na2HPO4 pH 7.0, 0.8% BSA, 5% SDS, 12% formamide 6 with 1/15-1/100 &mu;l of the purified, labeled reference DNA (2.4) per RNA sample. The amount of added reference DNA depends on how many different oligonucleotides are labeled (2.4). If there are over 100, then less dilution is needed.</li>
	<li>Dissolve the RNA pellet well with &tilde; 60 &mu;l of the hybridization solution.</li>
	<li>Add 20 &mu;l of water to each of the humidifying wells in the Corning microarray hybridization chamber base.</li>
	<li>Add the mixture of labeled RNA and reference DNA to the slide. Use a thin pipet tip, gently touch the edge of the lifterslip, and allow the solution to enter the space between the lifterslip and slide through suction.</li>
	<li>Place the hybridization chamber cover over the base, then seal the chamber with metal clips.</li>
	<li>Put the whole chamber inside the plastic bag that comes with the chamber cassette from Corning.</li>
	<li>Place the chamber cassette(s) inside a container with wetted paper towel; cover the container with plastic wrap and place it inside a 37&deg;C humid, cell culture incubator for &tilde; 24 hours. These precautions (3.6, 3.9, 3.10) ensure that the hybridization solution does not dry out during incubation.</li>
</ol>
4. Post-hybridization processing
<ol>
	<li>Disassemble the chamber cassettes one by one. Submerge a slide and its lifterslip in 2 x SSC at 22&deg;C. The lifterslip will naturally fall off the slide. Place it in 0.8 x SSC. Wash the slides in 0.8 x SSC twice, then three times in 0.4 x SSC, 1-2 min in total. Dry the slides using a centrifuge with a slide adapter. Wash the lifterslips with water and save for re-use later.</li>
	<li>Scan the slides with any suitable scanner, e.g., the Perkin Elmer ScanArray 5000 or Molecular Devices Axon GenePix 4000B microarray scanner. Scan at wavelengths close to 547 nm and 647 nm to obtain the corresponding image files.</li>
	<li>Use programs such as BlueFuse to quantify pixel intensities in the input, image files from 4.2). Inspect individual spots with the program to exclude abnormal spots on the microarrays from further consideration. These abnormal spots usually arise from poor slide printing or slide handling after hybridization.</li>
	<li>Use programs such as GeneSpring and Excel for data analysis and presentation. General considerations for microarray data normalization apply, which is beyond the scope of this paper.</li>
	<li>Once satisfactory signals are obtained after 4.3), strip the microarray slides for reuse 7. Rinse the slides in water, then immerse in pre-warmed, 1 mM NaOH and 0.1 x SSC in a staining dish at 62&deg;C for 10-20 minutes. Repeat the incubation once. Wash the slides a few times in water for up to 60 minutes with gentle shaking at 22&deg;C. Dry the slides using a centrifuge, and store at 22&deg;C.</li>
	<li>To use the regenerated slides, it is not necessary to pre-hybridize them again. Simply wash them in water followed by isopropanol, and dry the slides right before hybridization.</li>
</ol>
5. Representative Results:
We have largely followed the described procedures to profile global miRNA expression in thousands of samples, i.e., RNAs isolated from zebrafish to human specimen under many different conditions. Figure 1 shows a scanned image of a microarray to demonstrate very precise and strong hybridization signals on the slide. The Pearson correlation coefficients between technical replicates of microarray hybridization are &tilde; 0.99 7, indicating excellent reproducibility.
<img alt="Figure 1" src="/files/ftp_upload/3250/3250fig1.jpg" /> 
Figure 1 Composite image of a scanned miRNA microarray slide after hybridization. Red spots resulted from hybridizations by the reference DNA, green spots from the DY547-labelled RNA sample, while the yellow spots were from hybridizations by both the DNA and RNA to the same probes.

<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>

No conflicts of interest declared.

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 the same principles and procedures presented here can be used in custom microarray experiments to study gene families besides miRNAs. Of course, a major drawback about microarrays is that sequence information must be available a priori. This is not a problem for most protein gene families in model organisms, although how many miRNA genes there are remain unclear. The probe set we have been using was designed based on miRBase 8 Release 9.0 in October 2006. From recent studies 9, 10, it is apparent that the most abundant and most biologically relevant mammalian miRNAs are already covered by this probe set.
There are no major technical challenges to perform custom microarray experiments. Based on our experience, the keys to the success of a microarray experiment are: 1) to obtain well-printed slides, 2) to prepare RNA of good quality and in sufficient quantity, and 3) to properly wash the slides after hybridization. Trizol reagent typically yields sufficient amounts of intact RNA for subsequent studies. Nonetheless, miRNA expression varies among different tissues and cell lines. Under most conditions, 20-25 &mu;g of total RNA should give sufficiently strong microarray signals when labeled by the ligation method. Less RNA is required for miRNA-rich samples such as brain regions and neurons. If only 1-5 &mu;g of total RNA or less is available, other labeling methods may be used, e.g., RNA labeling kits from Invitrogen, and they are compatible with the custom microarray platform described here. For the best comparisons, one should always label at least one replicate of all the control and experimental samples simultaneously and hybridize them to the same print batch of slides in a single experiment. This research design will minimize the contribution to differential microarray signals resulting from the variations in sample labeling and processing and in slide quality. The reference DNA (in red channel) serves as a control for the quality of slide, hybridization, and washing. A successful hybridization will give clean and strong signals in red. If DNA signals are good but RNA signals are not, then one should trouble-shoot the steps of RNA isolation and labeling. For example, does the RNA have the ratio A260nm/A280nm between 1.9 and 2.1? Is the pellet reddish in 3.3?
Most of the investment for microarrays may be for slide printing and scanning. A microarray printer and a scanner are often equipped by the core facility at many universities and medical schools. Printing microarrays in bulk and reusing slides and lifterslips will significantly reduce the cost. Reusing slides has the additional advantage of improving the consistency of microarray experiments 7. We have reused slides more than six times and found the data quality still satisfactory. To ensure the best sensitivity for the most precious samples or samples with minimal input RNAs, however, it is still prudent to use fresh slides 7.

<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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19.5K Views.  University of Minnesota. 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.

Video: Performing Custom MicroRNA Microarray Experiments

Gibberella zeae Ascospore Production and Collection for Microarray Experiments.

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain quality. F. graminearum also causes stalk and ear rots of maize and is a producer of mycotoxins such as the trichothecenes that contaminate grain and are harmful to humans and livestock (Goswami and Kistler, 2004).

The fungus produces two types of spores. Ascospores, the propagules resulting from sexual reproduction, are the main source of primary infection.  These spores are forcibly discharged from mature perithecia and dispersed by wind (Francl et al 1999). Secondary infections are mainly caused by macroconidia which are produced by asexual means on the plant surface. To study the developmental processes of ascospores in this fungus, a procedure for their collection in large quantity under sterile conditions was required. Our protocol was filmed in order to generate the highest level of information for understanding and reproducibility; crucial aspects when full genome gene expression profiles are generated and interpreted. In particular, the variability of ascospore germination and biological activity are dependent on the prior manipulation of the material. The use of video for documenting every step in ascospore production is proposed in order to increase standardization, complying with the increasingly stringent requirements for microarray analysis. The procedure requires only standard laboratory equipment. Steps are shown to prevent contamination and favor time synchronization of ascospores.

To study the developmental processes of ascospores in Gibberella zeae, a procedure for collection under sterile conditions is filmed in order to generate the highest level of information for protocol description. This should facilitate the reproducibility of the experiment, a crucial aspect when full genome expression profile tests are implemented.

Gibberella zeae Produkcja Ascospore i Kolekcja dla Eksperymenty mikromacierzy.

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain ...

Preparation of Artificial Bilayers for Electrophysiology Experiments

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can be used for many different studies, such as the characterization of membrane-active peptides, the reconstitution of ion channels or investigations on how changes in lipid bilayer properties alter the function of bilayer-spanning channels.  Here, we show how to form a planar bilayer and how to isolate small patches from the bilayer, and in a second video  will also demonstrate a procedure for using gramicidin channels to determine changes in lipid bilayer elastic properties.   We also demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. Here, we demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can ...

Processing the Loblolly Pine PtGen2 cDNA Microarray

PtGen2 is a 26,496 feature cDNA microarray containing amplified loblolly pine ESTs. The array is produced in our laboratory for use by researchers studying gene expression in pine and other conifer species. PtGen2 was developed as a result of our gene discovery efforts in loblolly pine, and is comprised of sequences identified primarily from root tissues, but also from needle and stem.1,2 PtGen2 has been tested by hybridizing different Cy-dye labeled conifer target cDNAs, using both amplified and non-amplified indirect labeling methods, and also tested with a number of hybridization and washing conditions. This video focuses on the handling and processing of slides before and after pre-hybridization, as well as after hybridization, using some modifications to procedures developed previously.3,4 Also included, in text form only, are the protocols used for the generation, labeling and clean up of target cDNA s, as well as information on software used for downstream data processing. 
PtGen2 is printed with a proprietary print buffer that contains high concentrations of salt that can be difficult to remove completely. The slides are washed first in a warm SDS solution prior to pre-hybridization. After pre-hybridization, the slides are washed vigorously in several changes of water to complete removal of remaining salts. LifterSlips&trade; are then cleaned and positioned on the slides and labeled cDNA is carefully loaded onto the microarray by way of capillary action which provides for even distribution of the sample across the slide, and reduces the chance of bubble incorporation. Hybridization of targets to the array is done at 48&deg;C in high humidity conditions. After hybridization, a series of standard washes are done at 53&deg;C and room temperature for extended times. Processing PtGen2 slides using this technique reduces salt and SDS-derived artifacts often seen when the array is processed less rigorously. Hybridizing targets derived from several different conifer RNA sources, this processing protocol yielded fewer artifacts, reduced background, and provided better consistency among different experimental groups of arrays.

The cDNA microarray PtGen2 was developed for gene expression studies in loblolly pine, P. taeda, and other conifer species. Here, we show pre- and post-hybridization handling and washing techniques that can be used with this array to yield better consistency, reduced artifacts, and lower backgrounds.

PtGen2 is a 26,496 feature cDNA microarray containing amplified loblolly pine ESTs.  The array is produced in our laboratory for use by researchers ...

Global Gene Expression Analysis Using a Zebrafish Oligonucleotide Microarray Platform

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray technology is used in numerous biological disciplines in a variety of applications including global gene expression analysis in relation to developmental stage, to a disease state, and in toxic responses. Herein, we include a demonstration of global gene expression analysis using a comprehensive zebrafish-specific oligonucleotide microarray platform. The zebrafish expression microarray platform contains 385,000 probes, 60 base pairs in length, interrogating 37,157 targets with up to 12 probes per target. For this platform, all cDNA and genomic information available for the zebrafish was collected from various genomic databases including Ensembl (http://www.ensembl.org), VEGA (http://vega.sanger.ac.uk), UCSC (http://genome.ucsc.edu), and ZFIN (http://www.zfin.org). As a result this expression array provides complete coverage of the current zebrafish transcriptome. The zebrafish expression microarray was printed by Roche NimbleGen (Madison, WI). This technical demonstration includes the fluorescent labeling of a cDNA product, hybridization of the labeled cDNA product to the microarray platform, and array scanning for signal acquisition using the one color analysis strategy.  

Gene microarrays are powerful tools in gene expression profiling at a genome-wide level. This technology has application in a variety of biological disciplines including developmental biology and toxicology. In this video, we detail a protocol for global gene expression analysis using a comprehensive oligonucleotide microarray platform for the zebrafish.

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray ...

Measurement Of Neuromagnetic Brain Function In Pre-school Children With Custom Sized MEG

Magnetoencephalography is a technique that detects magnetic fields associated with cortical activity [1]. The electrophysiological activity of the brain generates electric fields  - that can be recorded using electroencephalography (EEG)- and their concomitant magnetic fields  - detected by MEG. MEG signals are detected by specialized sensors known as superconducting quantum interference devices (SQUIDs). Superconducting sensors require cooling with liquid helium at -270 &deg;C. They are contained inside a vacumm-insulated helmet called a dewar, which is filled with liquid. SQUIDS are placed in fixed positions inside the helmet dewar in the helium coolant, and a subject's head is placed inside the helmet dewar for MEG measurements. The helmet dewar must be sized to satisfy opposing constraints. Clearly, it must be large enough to fit most or all of the heads in the population that will be studied. However, the helmet must also be small enough to keep most of the SQUID sensors within range of the tiny cerebral fields that they are to measure. Conventional whole-head MEG systems are designed to accommodate more than 90% of adult heads. However adult systems are not well suited for measuring brain function in pre-school chidren whose heads have a radius several cm smaller than adults. The KIT-Macquarie Brain Research Laboratory at Macquarie University uses a MEG system custom sized to fit the heads of pre-school children. This child system has 64 first-order axial gradiometers with a 50 mm baseline[2] and is contained inside a magnetically-shielded room (MSR) together with a conventional adult-sized MEG system [3,4]. There are three main advantages of the customized helmet dewar for studying children. First, the smaller radius of the sensor configuration brings the SQUID sensors into range of the neuromagnetic signals of children's heads. Second, the smaller helmet allows full insertion of a child's head into the dewar. Full insertion is prevented in adult dewar helmets because of the smaller crown to shoulder distance in children. These two factors are fundamental in recording brain activity using MEG because neuromagnetic signals attenuate rapidly with distance. Third, the customized child helmet aids in the symmetric positioning of the head and limits the freedom of movement of the child's head within the dewar. When used with a protocol that aligns the requirements of data collection with the motivational and behavioral capacities of children, these features significantly facilitate setup, positioning, and measurement of MEG signals.

The advent of MEG systems sized for young children opens important new opportunities to study brain development. The new system, together with a protocol that aligns experimental requirements with the capacities of children, can be used to study cognitive and language processes in healthy, awake children aged three to six.

Magnetoencephalography is a technique that detects magnetic fields associated with cortical activity [1]. The electrophysiological activity of the brain ...

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

Genome Editing with CompoZr Custom Zinc Finger Nucleases (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such as chemical-based mutagenesis or random integration of DNA sequences confer indiscriminate genetic changes in an overall inefficient manner and require incorporation of undesirable synthetic sequences or use of aberrant culture conditions, potentially confusing biological study. By contrast, transient ZFN expression in a cell can facilitate precise, heritable gene editing in a highly efficient manner without the need for administration of chemicals or integration of synthetic transgenes.
Zinc finger nucleases (ZFNs) are enzymes which bind and cut distinct sequences of double-stranded DNA (dsDNA). A functional CompoZr ZFN unit consists of two individual monomeric proteins that bind a DNA "half-site" of approximately 15-18 nucleotides (see Figure 1). When two ZFN monomers "home" to their adjacent target sites the DNA-cleavage domains dimerize and create a double-strand break (DSB) in the DNA.1 Introduction of ZFN-mediated DSBs in the genome lays a foundation for highly efficient genome editing. Imperfect repair of DSBs in a cell via the non-homologous end-joining (NHEJ) DNA repair pathway can result in small insertions and deletions (indels). Creation of indels within the gene coding sequence of a cell can result in frameshift and subsequent functional knockout of a gene locus at high efficiency.2 While this protocol describes the use of ZFNs to create a gene knockout, integration of transgenes may also be conducted via homology-directed repair at the ZFN cut site.
The CompoZr Custom ZFN Service represents a systematic, comprehensive, and well-characterized approach to targeted gene editing for the scientific community with ZFN technology. Sigma scientists work closely with investigators to 1) perform due diligence analysis including analysis of relevant gene structure, biology, and model system pursuant to the project goals, 2) apply this knowledge to develop a sound targeting strategy, 3) then design, build, and functionally validate ZFNs for activity in a relevant cell line. The investigator receives positive control genomic DNA and primers, and ready-to-use ZFN reagents supplied in both plasmid DNA and in-vitro transcribed mRNA format. These reagents may then be delivered for transient expression in the investigator&#x2019;s cell line or cell type of choice. Samples are then tested for gene editing at the locus of interest by standard molecular biology techniques including PCR amplification, enzymatic digest, and electrophoresis. After positive signal for gene editing is detected in the initial population, cells are single-cell cloned and genotyped for identification of mutant clones/alleles.

Genome Editing with CompoZr Custom Zinc Finger Nucleases ZFNs

The CompoZr Custom Zinc-Finger Nuclease (ZFN) Service enables precise genome editing in any organism or cell line at any locus defined by the user. This article describes the process for the design, manufacture, validation and implementation of the CompoZr Custom ZFN Service.

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such ...

Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media

Stable miRNAs are present in all body fluids and some circulating miRNAs are protected from degradation by sequestration in small vesicles called exosomes. Exosomes can fuse with the plasma membrane resulting in the transfer of RNA and proteins to the target cell. Their biological functions include immune response, antigen presentation, and intracellular communication. Delivery of miRNAs that can regulate gene expression in the recipient cells via blood has opened novel avenues for target intervention. In addition to offering a strategy for delivery of drugs or RNA therapeutic agents, exosomal contents can serve as biomarkers that can aid in diagnosis, determining treatment options and prognosis.	Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.	
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media.	
 Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media

The presence of stable microRNAs (miRNAs) in exosomes has generated immense interest as a novel mode of intercellular communication, for their potential utility as biomarkers and as a route for therapeutic intervention. Here we demonstrate exosome purification from blood and culture media followed by quantitative PCR to identify miRNAs being transported.

Stable miRNAs are present in all body fluids and some circulating miRNAs  are protected from degradation by sequestration in small vesicles called  ...

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

Utilizing Custom-designed Galvanotaxis Chambers to Study Directional Migration of Prostate Cells

The physiological electric field serves specific biological functions, such as directing cell migration in embryo development, neuronal outgrowth and epithelial wound healing. Applying a direct current electric field to cultured cells in vitro induces directional cell migration, or galvanotaxis. The 2-dimensional galvanotaxis method we demonstrate here is modified with custom-made poly(vinyl chloride) (PVC) chambers, glass surface, platinum electrodes and the use of a motorized stage on which the cells are imaged. The PVC chambers and platinum electrodes exhibit low cytotoxicity and are affordable and re-useable. The glass surface and the motorized microscope stage improve quality of images and allow possible modifications to the glass surface and treatments to the cells. We filmed the galvanotaxis of two non-tumorigenic, SV40-immortalized prostate cell lines, pRNS-1-1 and PNT2. These two cell lines show similar migration speeds and both migrate toward the cathode, but they do show a different degree of directionality in galvanotaxis. The results obtained via this protocol suggest that the pRNS-1-1 and the PNT2 cell lines may have different intrinsic features that govern their directional migratory responses.

We present a method to apply a physiological electric field to migrating, immortalized prostate cells in a custom-made galvanotaxis chamber. Using this method, we demonstrate that 2 lines of non-tumorigenic prostate cells demonstrate different degrees of migration directionality in the field.

The physiological electric field serves specific biological functions, such as directing cell migration in embryo development, neuronal outgrowth and ...