Name: dna microarrays: sample quality control, array hybridization and scanning
Uploaded: 2011-03-15T11:00:00
Duration: 9 min 27 s
Description: Watch this Scientific Journal Video about DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning at JoVE.com

This work was supported by grants to E. D. from the James S. McDonnell Foundation 21stCentury Award Program, and a NIH Director's New Innovator Award. G. A. B. was supported in part by a postdoctoral fellowship from the California Institute of Regenerative Medicine.

1. Quality control analysis of RNA sample with 2100 Bioanalyzer
Before you start,
<ol>
	<li>Denature the ladder and your samples at 70 &deg;C for 10 min. Chill immediately on ice.</li>
	<li>Clean the instrument electrodes with RNaseZAP for 1 min, followed by RNase-free water for 10 sec. Allow the electrodes to air-dry.</li>
	<li>Prepare the gel matrix by pipeting 550 &mu;l of the gel matrix into a spin filter provided with the kit. Centrifuge at 1,500 RCF for 10 min at room temperature. Aliquot the filtered gel into 65 &mu;l aliquots and store at 4&deg;C for up to 1 month.</li>
	<li>To prepare the chip priming station,
	<ol class="lalpha">
		<li>Insert a 1-ml syringe through the clip and into the luer lock adapter.</li>
		<li>Adjust the base plate to position C by loosening the screw under the base, lifting the plate and retightening the screw.</li>
		<li>Adjust the syringe clip to the top position.</li>
	</ol>
	</li>
	<li>Equilibrate the dye concentrate to room temperature. Vortex for 10 sec. Add 1 &mu;l of dye to a 65 &mu;l aliquot of filtered gel. Vortex well and centrifuge at 13,000 RCF for 10 min at room temperature. Use within one day. 
	When you are ready to run your samples,</li>
	<li>Position a chip on the priming station. In this protocol we are using RNA 6000 nano chips. To load the gel,
	<ol class="lalpha">
		<li>Pipette 9 &mu;l of the gel-dye mix in the well marked with a white "G" against a black background. Make sure your tip is positioned at the very bottom of the well.</li>
		<li>Position the syringe plunger at 1 ml. Close the priming station. Make sure it click-locks.</li>
		<li>Press the plunger down slowly but steadily until it is held by the clip.</li>
		<li>Wait for 30 sec. Release the clip.</li>
		<li>Let the plunger go up by itself. After it stops moving, wait for a few seconds and pull the plunger back to the 1 ml position. Open the priming station.</li>
	</ol>
	</li>
	<li>Pipette 9 &mu;l of the gel-dye mix in each of the 2 wells marked "G".</li>
	<li>Pipette 5 &mu;l of the marker in each of the 12 sample wells and in the ladder well.</li>
	<li>Pipette 1 &mu;l of the prepared ladder in ladder well.</li>
	<li>Pipette 1 &mu;l of each sample into the sample wells.</li>
	<li>Pipette 1 &mu;l of the marker in each unused sample well.</li>
	<li>Vortex the chip for 1 min at 2,400 rpm.</li>
	<li>To run the chip,
	<ol class="lalpha">
		<li>Start the 2100 Expert Software.</li>
		<li>Position the chip and close the lid. If the instrument is on-line, an icon will show whether the lid is open or closed and what type of chip is inserted. Make sure the correct port is selected.</li>
		<li>Run the assay within 5 min of loading the samples to prevent evaporation.</li>
	</ol>
	</li>
</ol>
2. Amplification and labeling
To prepare for microarray analysis, RNA samples are amplified and labeled, usually in a reaction based on T7 RNA polymerase3. Double stranded cDNA is generated by reverse transcription. cDNA is used in an in vitro transcription reaction to generate what is known as cRNA. This reaction is performed in the presence of labeled ribonucleotides, producing microgram quantities of labeled RNA for array hybridization. The choice of amplification/labeling methods depends on the subsequent microarray platform to be used. In this section, we describe the generation of fluorescently labeled RNA using Agilent's Quick Amp labeling kit.
<ol>
	<li>Pipette 50 ng to 5 &mu;g of RNA in a 1.5 ml tube. The volume should not exceed 8.3 &mu;l. If necessary, concentrate your RNA sample by vacuum centrifugation or precipitation with alcohol.</li>
	<li>Add 1.2 &mu;l of T7 primer.</li>
	<li>Bring to a final volume of 11.5 &mu;l with RNase-free water.</li>
	<li>Incubate at 65&deg;C for 10 min to denature the RNA. We recommend using a thermocycler with a hot lid to prevent condensation in the top of the tube.</li>
	<li>During the 10 min incubation, warm 5X First Strand Buffer to 80&deg;C for 5 min. Vortex and spin down. Keep at RT until ready to use.</li>
	<li>After the 10 min incubation, quickly cool down the denatured RNA samples on ice.</li>
	<li>Prepare the cDNA master mix. Per reaction, add the following:
	<ol class="lalpha">
		<li>4 &mu;l 5X First Strand Buffer</li>
		<li>2 &mu;l 0.1 M DTT</li>
		<li>1 &mu;l 10 mM dNTPs</li>
		<li>1 &mu;l MMLV-RT enzyme</li>
		<li>0.5 &mu;l RNase Out.</li>
	</ol>
	</li>
	<li>Mix components by inverting the tube 4 times. Do not vortex. Spin down to collect the contents at the bottom of the tube.</li>
	<li>Add 8.5 &mu;l of master mix per sample. Mix by pipeting up and down.</li>
	<li>Incubate for 2 hr at 40&deg;C, followed by 10 min at 65&deg;C. We recommend using a thermocycler with a heated lid.</li>
	<li>Transfer the tubes to ice for 5 min.</li>
	<li>Prepare the transcription master mix. Per reaction, add the following:
	<ol class="lalpha">
		<li>15.3 &mu;l nuclease-free water</li>
		<li>20 &mu;l 4X transcription buffer</li>
		<li>6 &mu;l 0.1 M DTT</li>
		<li>8 &mu;l NTPs</li>
		<li>6.4 &mu;l 50% PEG (PEG should be prewarmed to 40&deg;C and vortexed to ensure resuspension)</li>
		<li>0.5 &mu;l RNase Out</li>
		<li>0.6 &mu;l inorganic pyrophosphatase</li>
		<li>0.8 &mu;l T7 RNA polymerase</li>
		<li>2.4 &mu;l Cy3- or Cy5 CTP</li>
	</ol>
	</li>
	<li>Briefly spin the samples to collect the contents at the bottom of the tube.</li>
	<li>Add 60 &mu;l of Transcription Master Mix per sample.</li>
	<li>Incubate at 40&deg;C for 2 hr.</li>
	<li>Add 20 &mu;l of RNase-free water.</li>
	<li>Purify the labeled cRNA to remove unincorporated nucleotides. We recommend using Qiagen's RNeasy mini columns.</li>
	<li>Quantify the labeled cRNA. We recommend using a NanoDrop2000 spectrophotometer in Microarray Measurement mode. Make sure you select RNA-40 as the sample type.</li>
	<li>Record sample concentration, yield (calculated as the concentration multiplied by the volume) and specific activity (calculated as 1000 multiplied by the ration of dye concentration over cRNA concentration). For microarray hybridization, it is necessary to achieve a yield of at least 825 ng and a specific activity of at least 8 pmol/&mu;g.</li>
</ol>
3. Microarray hybridization
<ol>
	<li>Turn on the hybridization station and set at 65&deg;C at least 2 hours before hybridization begins.</li>
	<li>To prepare the hybridization sample, mix the following in a 1.5 ml tube:
	<ol class="lalpha">
		<li>825 ng Cy3-cRNA (this is usually your "treated" sample)</li>
		<li>825 ng Cy5-cRNA (this is usually your "reference" sample)</li>
		<li>11 &mu;l 10X Blocking agent.</li>
		<li>Add RNase-free water to a final volume of 52.8 &mu;l</li>
		<li>Add 2.2 &mu;l fragmentation buffer.</li>
	</ol>
	</li>
	<li>Vortex at low speed to mix.</li>
	<li>Incubate at 60&deg;C for exactly 30 min. This is the cRNA fragmentation step, which generates labeled fragments for array hybridization. It is very important to incubate exactly as described. We recommend the use of a thermocycler with a heated lid. It is very important to incubate exactly as described to generate probes of the correct average length. Excessive or insufficient fragmentation will result in false negatives or false positives.</li>
	<li>Add 55 &mu;l of 2X Hybridization buffer to stop fragmentation. Mix by pipetting, taking special care not to introduce bubbles.</li>
	<li>Centrifuge for 1 min at maximum speed to collect the content at the bottom of the tube. If bubbles are observed, centrifuge longer to remove them.</li>
	<li>Place the tube on ice, protected from the light. Load the sample on the array as soon as possible.</li>
	<li>To load the array, first prepare the hybridization chamber. This protocol describes the use of Agilent's 4x44K arrays with the Roche-Nimblegen hybridization system and A4 mixer chambers.
	<ol class="lalpha">
		<li>Place a microarray slide in the assembly/disassembly tool, barcode side first.</li>
		<li>Open an A4 mixer and expose the adhesive. Holding the array and assembly/disassembly tool to prevent any movement, place the A4 mixer on the slide, starting at the far end. Make sure it is correctly aligned to the tool. Stick the mixer all along the array slide.</li>
		<li>Pull from the end of the mixer to remove the slide/mixer assembly.</li>
		<li>Using the braying tool, press all along the adhesive gasket to make sure it is tightly glued to theslide. Small air bubbles trapped between the adhesive and the slide can be seen against a dark background. Take extra time to remove these bubbles with the braying tool. Your array is now ready to be loaded.</li>
	</ol>
	</li>
	<li>Use a positive displacement pipette to load 100 &mu;l of the hybridization sample. Push the tip tightly inside the port hole; then dispense slowly and smoothly so that air is not trapped in the array area. Never try to suck the sample back up. Once the entire sample has been dispensed, keep the tip on the port hole without releasing the plunger. When the sample covers the whole array and liquid starts to come out the vent port, quickly remove the pipette. Only release the plunger once the tip is away from the slide.</li>
	<li>Gently dab the excess liquid at both ports. Make sure you do not draw sample out of the chamber itself.</li>
	<li>Cover the port holes with stickers by using tweezers.</li>
	<li>Put the slide on the hybridization station, and check that the bladder holes are correctly positioned over the O-rings. This will ensure proper mixing during hybridization.</li>
	<li>Close the slide cover and the station lid. Set the station to mixing mode B.</li>
	<li>Hybridize the array at 65&deg;C for 17 hours.</li>
	<li>Prepare Wash Buffer 2 by adding Triton X-102 to a final concentration of 0.005%. Keep at 37&deg;C overnight.</li>
</ol>
4. Microarray washing
<ol>
	<li>Add Triton X-102 to Wash Buffer 1 to a final concentration of 0.005%. Keep at room temperature.</li>
	<li>When hybridization is finished, fill a glass dish "A" with Wash Buffer 1. The dish should be big enough to hold the assembly/disassembly tool and allow some maneuvering as well. All washes should be done in glassware. Avoid plastic dishes, since they tend to leach compounds resulting in high background in the array.</li>
	<li>Put a slide rack and a stir bar in a staining dish "B". Fill with Wash buffer 1, making sure it covers the rack. Place on a stir plate at room temperature.</li>
	<li>Put a stir bar on an empty staining dish "C" and place on a stir plate.</li>
	<li>Remove the array and put it in the assembly/disassembly tool. Submerge the whole assembly in the dish "A".</li>
	<li>While holding the assembly/disassembly tool and the slide from opposite sides with one hand, carefully peel off the A4 mixer. Make sure you do not scratch the array area.</li>
	<li>Quickly place the slide in the rack in staining dish "B". From now on, exposure to the air should be minimized since the Cy5 dye is sensitive to ozone. Do not wash more than 8 arrays at a time.</li>
	<li>Wash for 1 minute stirring at medium speed.</li>
	<li>Fill staining dish "C" with pre-warmed Wash buffer 2. Transfer the slides and wash for 1 minute.</li>
	<li>Very slowly remove the slide rack from staining dish "C" to minimize droplets left behind on the slide.</li>
	<li>Dry the slide by spinning for 2 minutes. If a compatible centrifuge is not available, centrifuge the slide in a 50-ml conical tube or blow argon gas over it.</li>
	<li>Place slides in a 50-ml conical tube and fill it with argon gas. Scan immediately to avoid signal loss. We recommend use of the GenePix 4000B scanner from Molecular Devices.</li>
</ol>
5. Representative Results:
Good quality total RNA samples should produce only two major peaks when run in a Bioanalyzer for quality control, corresponding to the 2 major ribosomal RNA species. Some RNA degradation will show as a smear before the first ribosomal RNA peak. Severely degraded, low quality RNA will show a broad peak or a series of peaks at low retention times, while the 2 ribosomal RNA peaks will be of very low intensity or not identifiable at all. For examples of the 2100 Bioanalyzer output, see figure 1.
The Expert 2100 software will calculate an RNA Integrity Number, or RIN, as a quantitative measurement of RNA quality. High quality samples, with RIN values higher than 9, are obviously best for microarray applications. However, we have used samples with RIN values as low as 5.2 in generating microarray data of reasonable quality.
Good quality arrays should produce high signal at relatively low PMT values. In our experiment, most of the transcripts are expected to be present at similar levels in the experimental and reference sample; massive, widespread changes in gene expression will probably lack any biological significance. Therefore, most of the array should look yellow rather than green or red. Good quality signals should also be in a dynamic range such that the signal histograms fully overlap. For examples, see figure 2.
<img src="/files/ftp_upload/2546/2546fig1.jpg" alt="Figure 1" /> 
Figure 1. Example of four RNA samples isolated from human brain tumor tissue. RNA was isolated using the protocol presented in 3.1.1. Sample quality was assessed using the 2100 Bioanalyzer on an RNA 6000 chip as described in 3.1.3. Samples are representative of 4 distinct RNA qualities: A, very good RNA quality (RIN&gt;9); B, partially degraded RNA (RIN 5-6, note the significantly lower peak for 28S rRNA); C, highly degraded RNA (RIN&lt;3); D, almost completely degraded RNA (RIN=2). We have successfully used samples with qualities similar to or better than B (RIN&gt;5.2) for downstream microarray applications. Solid and open arrowheads indicate the position of the 18S and 28S rRNA species, respectively.
<img src="/files/ftp_upload/2546/2546fig2.jpg" alt="Figure 2" /> 
Figure 2. Examples of array images and scatter plots. A, whole slide preview, showing the four arrays in the 4X44K Agilent format; B, high resolution scan of one of the array areas, note that neither of the signals (red or green) is predominant; C, scatter plot of the image in B, note that the signals fully overlap and no more than 1x10-6 features are at saturating intensity.

<ol>
	<li>Diaz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+Decade+Later:+What+has+Gene+Expression+Profiling+Told+us+About+Neuronal+Cell+Types,+Brain+Function+and+Disease.">One Decade Later: What has Gene Expression Profiling Told us About Neuronal Cell Types, Brain Function and Disease.</a> Curr Genomics. 10, 318-318 (2009).</li><li>Barisone, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Expression profiling in Neuroscience, edited by Ioannis Karamanos. , (2010).</li><li>Gelder, R. N. V. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplified+RNA+synthesized+from+limited+quantities+of+heterogeneous+cDNA.">Amplified RNA synthesized from limited quantities of heterogeneous cDNA.</a> Proc Natl Acad Sci U S A. 87, 1663-1663 (1990).</li></ol>

No conflicts of interest declared.

Expression profiling with DNA microarrays provides a straightforward approach to identify differentially expressed genes between two biological samples. Successful expression profiling experiments require high quality RNA samples, robust labeling and hybridization. In our experience, the commercial arrays and labeling kits from Agilent provide high quality data at a reasonable cost. While Agilent also offers their own hybridization and scanning machinery, we favor the hybridization system from Roche-Nimblegen and the GenePix 4000B microarray scanner from Molecular Devices. Note that the Roche-Nimblegen hybridization unit was formerly known as the MAUI hybridization system. After the recent corporate acquisition by Roche, the A4 mixers have been discontinued. As of the moment of this writing, they can still be found through other sellers such as Kreatech Diagnostics (<a href="http://www.kreatech.com/">www.kreatech.com</a>; this website also offers a convenient array compatibility tool), but long-term stock availability is uncertain. Other hybridization systems are available (for example, from Agilent); however, if compatible mixers can be found for the array being used, we recommend the Roche-Nimblegen's system for its quality and reproducibility.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>2100 Bioanalyzer</td>
<td>&nbsp;</td>
<td>Agilent Technologies</td>
<td>G2943CA</td>
<td>Includes chip priming station, vortexer, software and laptop computer</td>
</tr>
<tr>
<td>2100 Bioanalyzer electrophoresis set</td>
<td>&nbsp;</td>
<td>Agilent Technologies</td>
<td>G2947CA</td>
<td>&nbsp;</td>
<tr>
<td>RNA 6000 Nano kit</td>
<td>&nbsp;</td>
<td>Agilent Technologies</td>
<td>5067-1511</td>
<td>includes size ladder, marker, gel matrix, dye, electrode cleaners, spin filters and nanochips</td>
</tr>
<tr>
<td>RNase Zap</td>
<td>&nbsp;</td>
<td>Applied Biosystems &#x2013; Ambion</td>
<td>AM9780M</td>
<td>&nbsp;</td>
<tr>
<td>Quick Amp labeling kit, two-color</td>
<td>&nbsp;</td>
<td>Agilent Technologies</td>
<td>5190-0444</td>
<td>&nbsp;</td>
<tr>
<td>RNeasy mini kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>74104</td>
<td>&nbsp;</td>
<tr>
<td>Gene Expression Hybridization Kit</td>
<td>&nbsp;</td>
<td>Agilent Technologies</td>
<td>5188-542</td>
<td>Includes Blocking agent, Fragmentation buffer and Hybridization buffer</td>
</tr>
<tr>
<td>Gene Expression Wash Buffer Kit</td>
<td>&nbsp;</td>
<td>Agilent Technologies</td>
<td>5188-5327</td>
<td>Includes Wash Buffers 1 and 2 and Triton X-102</td>
</tr>
<tr>
<td>Hybridization Station</td>
<td>&nbsp;</td>
<td>Roche Nimblegen</td>
<td>05223652001</td>
<td>4-slide model</td>
</tr>
<tr>
<td>Hybridization System Accesory Kit</td>
<td>&nbsp;</td>
<td>Roche Nimblegen</td>
<td>05327695001</td>
<td>Contains verification assembly for testing mixing, replacement O-rings, disassembly tool, forceps and brayer</td>
</tr>
<tr>
<td>A4 mixer hybridization chambers</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Whole Human Genome (4x44) Oligo Microarray</td>
<td>&nbsp;</td>
<td>Agilent Technologies</td>
<td>G4112F</td>
<td>&nbsp;</td>
<tr>
<td>Positive displacement pipette</td>
<td>&nbsp;</td>
<td>Gilson</td>
<td>F148504</td>
<td>&nbsp;</td>
<tr>
<td>Capillary pistons for positive displacement pipette</td>
<td>&nbsp;</td>
<td>Gilson</td>
<td>F148415</td>
<td>&nbsp;</td>
<tr>
<td>Microarray dryer</td>
<td>&nbsp;</td>
<td>Nimblegen</td>
<td>05223636001</td>
<td>&nbsp;</td>
<tr>
<td>Microarray fluorescent scanner, Axon GenePix 4000B</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>GENEPIX4000B</td>
<td>&nbsp;</td>
<tr>
<td>GenePix Pro 6.0 software</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

dna microarrays: sample quality control, array hybridization and scanning

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, different physiological or pathological states, response to therapy, and, in general, any condition that is being experimentally tested1. Expression profiling of neural tissues requires isolation of high quality RNA, amplification of the isolated RNA and hybridization to DNA microarrays. In this article we describe protocols for reproducible microarray experiments from brain tumor tissue2. We will start by performing a quality control analysis of isolated RNA samples with Agilent's 2100 Bioanalyzer "lab-on-a-chip" technology. High quality RNA samples are critical for the success of any microarray experiment, and the 2100 Bioanalyzer provides a quick, quantitative measurement of the sample quality. RNA samples are then amplified and labeled by performing reverse transcription to obtain cDNA, followed by in vitro transcription in the presence of labeled nucleotides to produce labeled cRNA. By using a dual-color labeling kit, we will label our experimental sample with Cy3 and a reference sample with Cy5. Both samples will then be combined and hybridized to Agilent's 4x44 K arrays. Dual-color arrays offer the advantage of a direct comparison between two RNA samples, thereby increasing the accuracy of the measurements, in particular for small changes in expression levels, because the two RNA samples are hybridized competitively to a single microarray. The arrays will be scanned at the two corresponding wavelengths, and the ratio of Cy3 to Cy5 signal for each feature will be used as a direct measurement of the relative abundance of the corresponding mRNA. This analysis identifies genes that are differentially expressed in response to the experimental conditions being tested.

Watch this Scientific Journal Video about DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning at JoVE.com

DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning

We demonstrate the use of DNA microarrays for expression profiling of the nervous system. We describe RNA quality control, sample labeling, and array hybridization and scanning.

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, ...

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Research

JoVE Journal

Biology

Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1. Recent advances in this technology have enabled high resolution comparison of whole genomes for the identification of genetic alterations in cancer and other genetic diseases 2. The Sub-Megabase Resolution Tiling-set array (or SMRT) array is comprised of a set of approximately thirty thousand overlapping bacterial artificial chromosome (BAC) clones that span the human genome in ~100 kilobase pair (kb) segments 2. These BAC targets are individually synthesized and spotted in duplicate on a single glass slide 2-4. Array CGH is based on the principle of competitive hybridization. Sample and reference DNA are differentially labeled with Cyanine-3 and Cyanine-5 fluorescent dyes, and co-hybridized to the array. After an incubation period the unbound samples are washed from the slide and the array is imaged. A freely available custom software package called SeeGH (www.flintbox.ca) is used to process the large volume of data collected - a single experiment generates 53,892 data points. SeeGH visualizes the log2 signal intensity ratio between the 2 samples at each BAC target which is vertically aligned with chromosomal position 5,6. The SMRT array can detect alterations as small as 50 kb in size 7. The SMRT array can detect a variety of DNA rearrangement events including DNA gains, losses, amplifications and homozygous deletions. A unique advantage of the SMRT array is that one can use DNA isolated from formalin fixed paraffin embedded samples. When combined with the low input requirements of unamplified DNA (25-100ng) this allows profiling of precious samples such as those produced by microdissection 7,8. This is attributed to the large size of each BAC hybridization target that allows the binding of sufficient labeled samples to produce signals for detection. Another advantage of this platform is the tolerance of tissue heterogeneity, decreasing the need for tedious tissue microdissection 8. This video protocol is a step-by-step tutorial from labeling the input DNA through to signal acquisition for the whole genome tiling path SMRT array.

This video is a technical demonstration of the hybridization protocol for whole genome tiling path array CGH, which scans the entire human genome using only 25-100 ng of DNA that can be isolated from a variety of sources, including archival formalin fixed material.

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1.  Recent ...

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

Fabrication and Use of MicroEnvironment microArrays (MEArrays)

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, distinct microenvironments can impose differentiation, migration, and proliferation phenotypes, and on the tissue level the microenvironment processes as complex as morphogenesis and tumorigenesis1. Not only do the cell and molecular contents of microenvironments impact the cells within, but so do the elasticity2 and geometry3 of the tissue. Defined as the sum total of cell-cell, -ECM, and -soluble factor interactions, in addition to physical characteristics, the microenvironment is complex. The phenotypes of cells within a tissue are partially due to their genomic content and partially due to the combinatorial interactions with the microenviroment. A major challenge is to link specific combinations of microenvironmental components with distinctive behaviours.
Here, we present the microenvironment microarray (MEArray) platform for cell-based functional screening of interactions with combinatorial microenvironments4. The method allows for simultaneous control of the molecular composition and the elastic modulus, and combines the use of widely available microarray and micropatterning technologies. MEArray screens require as few as 10,000 cells per array, which facilitates functional studies of rare cell types such as adult progenitor cells. A limitation of the technology is that entire tissue microenvironments cannot be completely recapitulated on MEArrays. However, comparison of responses in the same cell type to numerous related microenvironments, for instance pairwise combinations of ECM proteins that characterize a given tissue, will provide insights into how microenvironmental components elicit tissue-specific functional phenotypes.
MEArrays can be printed using a wide variety of recombinant growth factors, cytokines, and purified ECM proteins, and combinations thereof. The platform is limited only by the availability of specific reagents. MEArrays are amenable to time-lapsed analysis, but most often are used for end point analyses of cellular functions that are measureable with fluorescent probes. For instance, DNA synthesis, apoptosis, acquisition of differentiated states, or production of specific gene products are commonly measured. Briefly, the basic flow of an MEArray experiment is to prepare slides coated with printing substrata and to prepare the master plate of proteins that are to be printed. Then the arrays are printed with a microarray robot, cells are allowed to attach, grow in culture, and then are chemically fixed upon reaching the experimental endpoint. Fluorescent or colorimetric assays, imaged with traditional microscopes or microarray scanners, are used to reveal relevant molecular and cellular phenotypes (Figure 1).

Fabrication and Use of MicroEnvironment microArrays MEArrays

A combinatorial functional screening method for gaining insights into the impacts of the molecular composition of microenvironments on cellular functions is described. The method takes advantage of existing microarray-based technologies to generate arrays of defined combinatorial microenvironments that support cell adhesion and functional analysis.

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, ...

Array Comparative Genomic Hybridization (Array CGH) for Detection of Genomic Copy Number Variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a clinical diagnostic service laboratory. DNA extracted from a patient&#8217;s sample (blood, saliva or other tissue types) is labeled with a fluorochrome (either cyanine 5 or cyanine 3). A reference DNA sample is labeled with the opposite fluorochrome. There follows a cleanup step to remove unincorporated nucleotides before the labeled DNAs are mixed and resuspended in a hybridization buffer and applied to an array comprising ~60,000 oligonucleotide probes from loci across the genome, with high probe density in clinically important areas. Following hybridization, the arrays are washed, then scanned and the resulting images are analyzed to measure the red and green fluorescence for each probe. Software is used to assess the quality of each probe measurement, calculate the ratio of red to green fluorescence and detect potential copy number variants.

Array Comparative Genomic Hybridization Array CGH for Detection of Genomic Copy Number Variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a diagnostic service laboratory.

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its ...

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly ...

Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state by transmission electron microscopy (TEM). This can be achieved with very low quantity of protein and in the buffer of choice, without the use of any stain, which is very useful to determine structure-function correlations of macromolecules. When combined with single-particle image processing, the technique has found widespread usefulness for 3D structural determination of purified macromolecules. 
The protocol presented here explains how to perform cryoEM and examines the causes of most commonly encountered problems for rational troubleshooting; following all these steps should lead to acquisition of high quality cryoEM images. The technique requires access to the electron microscope instrument and to a vitrification device. Knowledge of the 3D reconstruction concepts and software is also needed for computerized image processing. Importantly, high quality results depend on finding the right purification conditions leading to a uniform population of structurally intact macromolecules. 
The ability of cryoEM to visualize macromolecules combined with the versatility of single particle image processing has proven very successful for structural determination of large proteins and macromolecular machines in their near-native state, identification of their multiple components by 3D difference mapping, and creation of pseudo-atomic structures by docking of x-ray structures. The relentless development of cryoEM instrumentation and image processing techniques for the last 30 years has resulted in the possibility to generate de novo 3D reconstructions at atomic resolution level.

Do&#039;s and Don&#039;ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

This paper describes the cryo-electron microscopy methodology, which is used to obtain high quality microscopic images of macromolecules in their near-native state. The method yields images suitable for further computerized processing using the single particle approach, devised to generate the 3D reconstruction of a macromolecule.

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

Synthesis of Wavelength-shifting DNA Hybridization Probes by Using Photostable Cyanine Dyes

In this protocol, we demonstrate a method for the synthesis of 2&#39;-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase synthesis using standard phosphoramidite chemistry. Oligonucleotides are post-synthetically labeled by two new photostable cyanine dyes using copper-catalyzed click-chemistry. The synthesis of both donor and acceptor dye is described and is performed in three consecutive steps. With the DNA as the surrounding architecture, these two dyes undergo an energy transfer when they are brought into close proximity by hybridization. Therefore, annealing of two single stranded DNA strands is visualized by a change of fluorescence color. This color change is characterized by fluorescence spectroscopy but can also be directly observed by using a handheld ultraviolet (UV) lamp. The concept of a dual fluorescence color readout makes these oligonucleotide probes excellent tools for molecular imaging especially when the described photostable dyes are used. Thereby, photobleaching of the imaging probes is prevented, and biological processes can be observed in real time for a longer time period.

Photostable cyanine dyes are attached to oligonucleotides to monitor hybridization by energy transfer.

In this protocol, we demonstrate a method for the synthesis of 2&#39;-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase ...

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we showed that D. discoideum is remarkably resilient to protein aggregation and can be used to gain insights into the cellular protein quality control system. However, the use of D. discoideum as a model system poses several challenges to microscopy-based experimental approaches, such as the high motility of the cells and their susceptibility to photo-toxicity. The latter proves to be especially challenging when studying protein homeostasis, as the phototoxic effects can induce a cellular stress response and thus alter to behavior of the protein quality control system. 
Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time.

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The social amoebae Dictyostelium discoideum has recently been established as a system to study protein misfolding and proteostasis. Here, we describe a new imaging-based methodology to study temperature-induced protein aggregation and the cellular stress response in D. discoideum.

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we ...

Array Tomography Workflow for the Targeted Acquisition of Volume Information using Scanning Electron Microscopy

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission Electron Microscopy (TEM) provided insight into cell ultrastructure, but in the recent decade, the development of modern Scanning Electron Microscopes (SEM) has changed the way of looking inside the cells. Even though the resolution of TEM is superior when protein-level structural details are needed, SEM-resolution is sufficient for the majority of the organelle-level cell biology-related questions. The advancement in technology enabled automatic volume acquisition solutions such as Serial block-face imaging (SBF-SEM) and Focused ion beam SEM (FIB-SEM). Nevertheless, to this day, these methods remain inefficient when the identification and navigation to areas of interest are crucial. Without the means for precise localization of target areas before imaging, operators need to acquire much more data than they need (in SBF-SEM), or, even worse, prepare many grids and image them all (in TEM). We propose the strategy of "lateral screening" using Array Tomography in SEM, which facilitates the localization of areas of interest, followed by automated imaging of the relevant fraction of the total sample volume. Array tomography samples are conserved during imaging, and they can be arranged into section libraries ready for repeated imaging. Several examples are shown in which lateral screening enables us to analyze structural details that are incredibly challenging to access with any other method.

We describe the preparation of ribbons of serial sections and their collection on large transfer support for use as Array Tomography samples, along with automated imaging procedures in a scanning electron microscope. The protocol allows screening, retrieval, and targeted imaging of local, rare events, and the acquisition of large data volumes.

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission ...