The labeling, hybridization, and washing protocols herein contain some modifications to those developed previously by Dr. J. Quackenbush while at The Institute for Genomic Research (TIGR), Dr. Shawn Levy at the Vanderbilt Microarray Shared Resource (VMSR), and Dr. Rob Alba while at the Boyce Thompson Institute, (BTI) Cornell University.

(Note Sections 1 - 4 Not Demonstrated in Video)
Preparing Cy-Labeled Target cDNA
What follows is the protocol we use for cDNA synthesis from loblolly pine total RNA and indirect cDNA labeling prior to microarray hybridizations. Although a few non-trivial modifications have been made, the protocol below is similar to that in the instruction manual provided with the Invitrogen&#8217;s SuperScript Indirect cDNA Labeling Kit.
Part 1: First-Strand cDNA Synthesis Reaction
<ol>
	<li>Mix and briefly spin each kit component before use.</li>
	<li>Prepare reactions as follows:
	<ul>
		<li>X&#181;l DEPC-H2O</li>
		<li>X&#181;l total RNA, 20 &#181;g/reaction (pretreated with Ambion&#8217;s Turbo DNase)</li>
		<li>2&#181;l Anchored Oligo(dT)20 Primer (2.5&#181;g/&#181;l)</li>
		<li>1&#181;l Random Hexamer Primer</li>
		<li style="list-style:none;">Total Volume = 18&#181;l</li>
	</ul>
	</li>
	<li>Incubate tubes at 70&#176;C for 5 minutes, and then place on ice for 1-2 minutes.</li>
	<li>Add the following to each reaction tube on ice:
	<ul>
		<li>6&#181;l 5X First-Strand buffer</li>
		<li>1.5&#181;l 0.1M DTT</li>
		<li>1.5&#181;l 10mM dNTP mix</li>
		<li>1&#181;l RNaseOUT (40U/&#181;l)</li>
		<li>2&#181;l SuperScript III RT (400U/&#181;l)</li>
	</ul>
	</li>
	<li>Mix gently and spin briefly. Incubate tube overnight at 45&#176;C.</li>
	<li>Add 15&#181;l of 1M NaOH to each reaction tube and mix thoroughly.</li>
	<li>Incubate tube at 70&#176;C for 10 minutes.</li>
	<li>Add 15&#181;l of 1M HCl; mix gently.</li>
	<li>Add 20&#181;l 3M sodium acetate (pH 5.2); mix gently.</li>
</ol>
Part 2: Purifying First-Strand cDNA.
<ol>
	<li>Add 500&#181;l of Loading Buffer to the cDNA (from Step 1.9) and mix well.</li>
	<li>Place a SNAP Purification Column (supplied with the Indirect Labeling kit) on a collection tube and load your cDNA on the column.</li>
	<li>Spin at 14,000g at room temperature for 1 minute; discard the flow-through.</li>
	<li>Place the SNAP Column onto the same collection tube and add 500&#181;l of Wash Buffer.</li>
	<li>Spin at 14,000g at room temperature for 1 minute; discard the flow-through.</li>
	<li>Repeat Steps 2.4 and 2.5 three times, for a total of three 500&#181;l washes.</li>
	<li>Spin one more time at 14,000g at room temperature for 1 minute sec; discard the flow-through.</li>
	<li>Place the SNAP Column onto a new 1.5ml tube.</li>
	<li>Add 50&#181;l of warm DEPC-water (50&#176;C) to the SNAP Column and incubate column at room temp for 1 minute. Elute the cDNA via a spin at 14,000g at room temp for 1 minute.</li>
	<li>Repeat Step 2.9, using 100&#181;l of warm DEPC-water and the same 1.5ml tube for the eluent.</li>
	<li>Add 20&#181;l of 3M sodium acetate (pH 5.2) to the eluent from steps 2.9 and 2.10.</li>
	<li>Add 3&#181;l of glycogen (20mg/ml) to the tube and mix.</li>
	<li>Add 500&#181;l of ice-cold 100% EtOH, mix well, and incubate the tube for 1 hour at &#8211;80&#176;C.</li>
	<li>Spin the tube at 14,000g at 4&#176;C for 20 minutes. Carefully remove the supernatant.</li>
	<li>Add 500&#181;l of ice-cold 70% EtOH and spin the tube at 14,000g at 4&#176;C for 5 minute. Carefully remove the supernatant.</li>
	<li>Air-dry the sample for 5 minutes; ensure that all EtOH is removed.</li>
	<li>Warm the 2X Coupling Buffer at 37&#176;C for 5 minutes and re-suspend the cDNA sample in 5&#181;l of warm 2X Coupling Buffer.</li>
	<li>Heat the cDNA/Coupling buffer at 50&#176;C for 10 minutes and vortex well. Ensure that your cDNA pellet is fully re-suspended in the 2X Coupling Buffer.</li>
</ol>
Part 3: Labeling with Fluorescent Dye
When preparing the reaction, be careful to minimize exposure of the dye solution to light. Also, DMSO is hygroscopic and will absorb moisture from the air, which will react with the NHS ester of the dye and significantly reduce the coupling reaction efficiency. Keep the DMSO supplied in the kit in an amber screw-capped vial at &#8211;20&#176;C, and let the vial warm to room temperature before opening to prevent condensation. Use only the DMSO provided with this kit.
<ol>
	<li>Open one packet of Cy-3 or Cy-5 Dye and add 75&#181;l of DMSO directly to the dye vial.</li>
	<li>Add 5&#181;l of the DMSO/dye solution to the tube from Step 2.18.</li>
	<li>Mix well and incubate the tube at room temperature in the dark for 1 hour.</li>
	<li>Add 20&#181;l of 3M Sodium Acetate (pH 5.2) to the dye-coupled cDNA solution.</li>
	<li>Add 500&#181;l of Loading Buffer to the cDNA solution. Mix well by vortexing.</li>
	<li>Place a SNAP Column onto a clear collection tube and load the cDNA/buffer solution.</li>
	<li>Spin at 14,000g at room temperature for 1 minute; discard the flow-through.</li>
	<li>Place the SNAP Column on the same collection tube; add 500&#181;l of Wash Buffer to column.</li>
	<li>Spin at 14,000g at room temperature for 1 minute; discard the flow-through.</li>
	<li>Repeat Steps 3. 8-3.9 three times, for a total of three 500&#181;l washes.</li>
	<li>Spin one more time at 14,000g at room temperature for 1 minute; discard the flow-through.</li>
	<li>Place the SNAP Column onto a new amber collection tube.</li>
	<li>Add 65&#181;l of warm DEPC-water (50&#176;C) to the SNAP Column and incubate at room temperature for 1 minute.</li>
	<li>Spin at 14,000g at room temperature for 1 minute and collect the flow-through. The flow-through should contain 60&#181;l of your purified dye-coupled cDNA.</li>
</ol>
Part 4: Assessing the Labeling Procedure.
<ol>
	<li>Conduct a spectrophotometer assay to assess each Cy-cDNA labeling. Calculate the total number of pmol of synthesized cDNA: [OD260 x vol. x 37ng/&#956;l x 103ng/pg]/324.5 pg/pmol. Calculate the total pmol of incorporated Cy-Dye for each labeling reaction: pmol Cy3 = [OD550 X vol.]/0.15; pmol Cy5 = [OD650 X vol.]/0.25. Next, calculate the pmol of nucleotide/pmol dye ratio for each reaction. Optimal reactions generate &#62;6000pmol of cDNA (per 60ul), &#62;200pmol of Cy-Dye (per 60ul), and a nucleotide/dye ratio that is &#60;30.</li>
</ol>
Part 5: Slide Pre-Wash
<ol>
	<li>The PtGen2 Array is printed on Corning UltraGaps slides (amino-silane coated) and cDNAs are UV-cross linked. Print spots are clearly visible due to the high salt content of the buffer.</li>
	<li>Use a diamond tip pen to mark the slide for the upper and lower areas of the print area (optional).</li>
	<li>Put approximately 250mL pre-warmed to 43&#176;C 0.2% SDS solution into microscope slide wash dish (3&#8221; x 4&#8221;).</li>
	<li>Place slides to be pre-hybridized in the slide rack and plunge into the SDS solution 15-20 times very vigorously to remove the salts on the slides.</li>
	<li>Using forceps, carefully remove the slides from the rack and place them in a Coplin jar containing Pre-hybridization Buffer (5X SSC, 0.1% SDS, 1% BSA) that has been pre-warmed to 43&#176;C. Incubate at 43&#176;C for at least one hour.</li>
</ol>
Part 6: Pre-Hybridization
<ol>
	<li>Set up five slide washing dishes, each filled with millipure dH2O, and one Coplin jar filled with molecular grade isopropanol. Place the slides in a slide rack and process sequentially through five dishes by dunking 20 times each.</li>
	<li>Remove slides two at a time from the fifth water wash and dip 5-6 times in the isopropanol and immediately place in slides into the slide microfuge (Chipmate benchtop, 2 positions) and spin 1 minute to dry.</li>
	<li>Clean slides with compressed air filtered through a 0.22 &#956;M filter cartridge and place them with the bar code facing up in a Genomic Solutions Hybridization Chamber.</li>
	<li>Clean LifterSlips&#8482; with filtered air and position them on the slides with the white strips facing down. Use a yellow pipet tip to gently align the lifter slip on the slide (Washing LifterSlips&#8482; first in 70% ethanol is optional. We have found it to be unnecessary.)</li>
</ol>
Part 7: Hybridization
Should be carried out in the dark or low light.
<ol>
	<li>For each slide to be hybridized, calculate the amount of Cy-5 and Cy-3 labeled cDNA to give 50-75pmol of each target. Combine those amounts into a single tube and dry in a vacuum desiccator with the heat setting at 37-45&#176;C. Avoid drying completely!</li>
	<li>Resuspend the dried probes in 60mL hybridization buffer (made fresh that day) and vortex briefly.</li>
	<li>Incubate the resuspended cDNAs for 5 minutes in a 95&#176;C bath and then quick microfuge for 30 seconds.</li>
	<li>Load the entire resuspended probe volume by slowly pipetting at the slide-LifterSlip&#8482; junction at the bottom (bar code end) of the slide. The slide will load by way of capillary action. (Some prefer to pipette on to the slide and then gently overlay the LifterSlip&#8482; this is a one shot method since if you get bubbles you cannot move the LifterSlip&#8482; after placement which can lead to spot smearing artifacts)</li>
	<li>Place 20uL of 100mM DTT into each humidity well located at the ends of the chamber.</li>
	<li>Place the top on the chamber and tighten the cam screws. Cover the chamber with aluminum foil and immerse into a 48&#176;C water bath for 12-18 hours with gentle agitation at 50-60 rpm.</li>
</ol>
Part 8: Post-Hybridization Washes
All washes should be carried out in the dark or low light. Slides should never be allowed to dry until the final step.
<ol>
	<li>Set up one Coplin jar filled with Wash Solution #1 @ 53&#176;C, and four slide washing dishes filled with 250mL of each of the following: 1) Wash Solution #1 @ 53&#176;C , 2) Wash Solution #2 @ room temperature, 3 &#38; 4) two dishes containing Wash Solution #3 @ room temperature.</li>
	<li>Carefully remove the slides from the hybridization chambers and place them two at a time into the Coplin jar. Leave for 30 seconds, then lift the slide out slowly and the lifter slip will remain in the jar. Place slides in the slide rack and immediately immerse into Wash Solution #1 (1X SSC, 0.2% SDS, preheat to 43&#176;C). Repeat until all LifterSlips are removed and all slides to be washed are in the Wash Solution #1 dish. Plunge rack up and down 10 times vigorously.</li>
	<li>Place dish in 53&#176;C in air shaker with shaking at 50-100 rpm for 10 minutes.</li>
	<li>Transfer rack to Wash Solution #2 (0.1X SSC, 0.2% SDS), plunge 10 times, and incubate at room temperature, slow shaking for 10 minutes.</li>
	<li>Dip rack 15 times into the first Wash Solution #3 (0.1X SSC) to remove any residual SDS carry over from the Wash Solution #2. Place rack in the second Wash Solution #3 dish, plunge 10 times, and then slow shaking for 10 minutes.</li>
	<li>Place slides in 50mL Falcon capped tubes and spin @ 1,500 rpm in centrifuge equipped with a swinging bucket rotor. Remove slides to light proofed container, we use another Falcon tube covered with aluminum foil, and purge air from the tube with nitrogen gas, cap tightly (this reduces oxidation of the Cy dyes, especially Cy-5 in the summer when ozone levels are highest). Slides can be stored for a few hours prior to scanning in the nitrogen purged tubes; however, best signal results are obtained when scanning is done immediately after washing.</li>
</ol>
Part 9: Data Collection and Statistical Analysis
<ol>
	<li>Scan image data is collected on a PerkinElmer ScanArray. We use a 10 micron scan setting and balance Cy3 and Cy5 signals using the line adjustment method as per the manufacturer&#8217;s instructions.</li>
	<li>TIF files are gridded and raw signal data collected and filtered with ImaGene Version 7.5 (BioDiscovery, Inc., El Segundo, CA)</li>
	<li>Data normalization and evaluation to determine statistically valid differentially expressed species is done with BRB Array tools Ver. 3.7.</li>
	<li>Pattern analysis to facilitate determination of coordinately expressed genes is done with Gene Expression Pattern Analysis Software (GEPAS) Ver. 3.1.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/1182/1182fig1.jpg" />
Figure 1. Example of PtGen2 array processed using this protocol.
<img alt="Figure 2" src="/files/ftp_upload/1182/1182fig2.jpg" />
Figure 2. Example of PtGen2 array processed using standard hybridization and washing protocols.

<ol>
	<li>Lorenz, W. W., Sun, F., Liang, C., Kolychev, D., Wang, H., Zhao, X., Cordonnier-Pratt, M. M., Pratt, L. H., Dean, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Water+stress-responsive+genes+in+loblolly+pine+(Pinus+taeda)+roots+identified+by+analyses+of+expressed+sequence+tag+libraries.">Water stress-responsive genes in loblolly pine (Pinus taeda) roots identified by analyses of expressed sequence tag libraries.</a> Tree Physiol. 26, 1-16 (2006).</li><li>Lorenz, W. W., Dean, J. F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> unpublished data. , .</li><li>Hegde, P., Qi, R., Abernathy, K., Gay, C., Dharap, S., Gaspard, R., Hughes, J. E., Snesrud, E., Lee, N., Quackenbush, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+concise+guide+to+cDNA+microarray+analysis.">A concise guide to cDNA microarray analysis.</a> Biotechniques. 29, 548-562 (2000).</li><li>Alba, R., Fei, Z., Payton, P., Liu, Y., Moore, S. L., Debbie, P., Cohn, J., D'Ascenzo, M., Gordon, J. S., Rose, J. K. C., Martin, G., Tanksley, S. D., Bouzayen, M., Molly, M., Jahn, M. M., Giovannoni, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ESTs,+cDNA+microarrays,+and+gene+expression+profiling:+tools+for+dissecting+plant+physiology+and+development.">ESTs, cDNA microarrays, and gene expression profiling: tools for dissecting plant physiology and development.</a> The Plant Journal. 39, 697-714 (2004).</li><li>Lorenz, W. W., Simões, M., Miguel, C., Dean, J. F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Gene+Expression+changes+in+Pinus+species+using+a+Loblolly+pine+cDNA+microarray.">Analysis of Gene Expression changes in Pinus species using a Loblolly pine cDNA microarray.</a> IUFRO-CTIA 2008 Joint Conference. , (2008).</li><li>Simões, M., Lorenz, W. W., Alba, A., Gonçalves, S., Dean, J., Miguel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+gene+expression+analysis+during+P.+pinaster+embryo+development.">Global gene expression analysis during P. pinaster embryo development.</a> 7th Plant Genomics European Meeting. , (2008).</li></ol>

PtGen2 is a recently developed, custom cDNA microarray that was designed to be used primarily by the loblolly pine research community. Preliminary results generated thus far have shown the array to be a valuable tool in the evaluation of transcriptional events that occur in response to drought stress, as well as in monitoring changes that occur during embryo development in maritime pine, Pinus pinaster 5,6 We have also recently demonstrated that PtGen2 works well in cross species hybridizations using...

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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>Pre-hybridization Buffer</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>5X SSC, 0.1% SDS, 1% BSA</td>
</tr>
<tr>
<td>Hybridization Buffer</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>30% formamide, 5X SSC, 5X Denhardt&#x2019;s, 1% PolyA RNA (Invitrogen, POLYA.GF), 0.1% SDS</td>
</tr>
<tr>
<td>Wash Solution #1</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1X SSC, 0.2% SDS, preheat to 43&deg;C</td>
</tr>
<tr>
<td>Wash Solution #2</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>0.1X SSC, 0.2% SDS</td>
</tr>
<tr>
<td>Wash Solution #3</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>0.1X SSC</td>
</tr>
<tr>
<td>Isopropyl Alcohol</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>34959-2.5L</td>
<td>&nbsp;</td>
<tr>
<td>50mL Conical Tubes</td>
<td>Other</td>
<td>Falcon&#x2122;</td>
<td>352070</td>
<td>&nbsp;</td>
<tr>
<td>15mL Conical Tubes</td>
<td>Other</td>
<td>Falcon&#x2122;</td>
<td>352196</td>
<td>Use this or a similar cap placed at the bottom of each 50mL conical tube during centrifugation</td>
</tr>
<tr>
<td>Coplin Jar</td>
<td>&nbsp;</td>
<td>Wheaton&#x2122;</td>
<td>900520</td>
<td>&nbsp;</td>
<tr>
<td>Staining Dish & Rack </td>
<td>Other</td>
<td>Wheaton&#x2122;</td>
<td>900200</td>
<td>&nbsp;</td>
<tr>
<td>Albumin from bovine serum (BSA)</td>
<td>Other</td>
<td>Sigma&#x2122;</td>
<td>A-9418</td>
<td>&nbsp;</td>
<tr>
<td>PolyA RNA</td>
<td>&nbsp;</td>
<td>Invitrogen&#x2122;</td>
<td>POLYA.GF</td>
<td>&nbsp;</td>
<tr>
<td>SuperScriptTM Indirect cDNA Labeling </td>
<td>Kit</td>
<td>Invitrogen&#x2122;</td>
<td>L1014-02</td>
<td>&nbsp;</td>
<tr>
<td>Turbo DNase</td>
<td>Kit</td>
<td>Ambion</td>
<td>AM1907</td>
<td>&nbsp;</td>
<tr>
<td>System</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cy-5 Dye</td>
<td>Reagent</td>
<td>GE&#x2122;</td>
<td>PA25001</td>
<td>&nbsp;</td>
<tr>
<td>Cy-3 Dye</td>
<td>Reagent</td>
<td>GE&#x2122;</td>
<td>PA23001</td>
<td>&nbsp;</td>
<tr>
<td>LifterSlips&trade;</td>
<td>Other</td>
<td>Erie Scientific Company&#x2122; </td>
<td>25X601</td>
<td>&nbsp;</td>
<tr>
<td>HybChambers</td>
<td>Equipment</td>
<td>GeneMachines</td>
<td>JHYB200003</td>
<td>&nbsp;</td>		</tbody>
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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.

Watch this Scientific Journal Video about Processing the Loblolly Pine PtGen2 cDNA Microarray at JoVE.com

Processing the Loblolly Pine PtGen2 cDNA Microarray

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 ...

processing-the-loblolly-pine-ptgen2-cdna-microarray

Research

JoVE Journal

Biology

7.3K Views.  University of Georgia (UGA). 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.

Video: Processing the Loblolly Pine PtGen2 cDNA Microarray

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 ...

The Microfluidic Probe: Operation and Use for Localized Surface Processing

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. Microfluidics is commonly associated with closed microchannels which limit their use to samples that can be introduced, and cultured in the case of cells, within a confined volume. On the other hand, micropipetting system have been used to locally perfuse cells and surfaces, notably using push-pull setups where one pipette acts as source and the other one as sink, but the confinement of the flow is difficult in three dimensions. Furthermore, pipettes are fragile and difficult to position and hence are used in static configuration only. 
The microfluidic probe (MFP) circumvents the constraints imposed by the construction of closed microfluidic channels and instead of enclosing the sample into the microfluidic system, the microfluidic flow can be directly delivered onto the sample, and scanned across the sample, using the MFP. . The injection and aspiration openings are located within a few tens of micrometers of one another so that a microjet injected into the gap is confined by the hydrodynamic forces of the surrounding liquid and entirely aspirated back into the other opening. The microjet can be flushed across the substrate surface and provides a precise tool for localized deposition/delivery of reagents which can be used over large areas by scanning the probe across the surface. 
In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer.

In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer solution.

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. ...

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications.  It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample.  Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated.  The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.    

The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

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 ...

Digital Microfluidics for Automated Proteomic Processing

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from extremely heterogeneous fluids (i.e. serum, whole blood, etc.) and (ii) extensive biochemical processing prior to analysis. Here, we report a new digital microfluidics (DMF) based method integrating several processing steps used in clinical proteomics. This includes protein extraction, resolubilization, reduction, alkylation and enzymatic digestion. Digital microfluidics is a microscale fluid-handling technique in which nanoliter-microliter sized droplets are manipulated on an open surface. Droplets are positioned on top of an array of electrodes that are coated by a dielectric layer - when an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric. The charges serve as electrostatic handles that can be used to control droplet position, and by biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface. Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays. This represents a significant advance over conventional methods (relying on manual pipetting or robots), and has the potential to be a useful new tool in clinical proteomics.
Mais J. Jebrail, Vivienne N. Luk, and Steve C. C. Shih contributed equally to this work.
Sergio L. S. Freire's current address is at the University of the Sciences in Philadelphia located at 600 South 43rd Street, Philadelphia, PA 19104.

Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and ...

An Improved Method of RNA Isolation from Loblolly Pine (P. taeda L.) and Other Conifer Species

Tissues isolated from conifer species, particularly those belonging to the Pinaceae family, such as loblolly pine (Pinus taeda L.), contain high concentrations of phenolic compounds and polysaccharides that interfere with RNA purification. Isolation of high-quality RNA from these species requires rigorous tissue collection procedures in the field and the employment of an RNA isolation protocol comprised of multiple organic extraction steps in order to isolate RNA of sufficient quality for microarray and other genomic analyses. The isolation of high-quality RNA from field-collected loblolly pine samples can be challenging, but several modifications to standard tissue and RNA isolation procedures greatly improve results. The extent of general RNA degradation increases if samples are not properly collected and transported from the field, especially during large-scale harvests. Total RNA yields can be increased significantly by pulverizing samples in a liquid nitrogen freezer mill prior to RNA isolation, especially when samples come from woody tissues. This is primarily due to the presence of oxidizing agents, such as phenolic compounds, and polysaccharides that are both present at high levels in extracts from the woody tissues of most conifer species. If not removed, these contaminants can carry over leading to problems, such as RNA degradation, that result in low yields and a poor quality RNA sample. Carryover of phenolic compounds, as well as polysaccharides, can also reduce or even completely eliminate the activity of reverse transcriptase or other polymerases commonly used for cDNA synthesis. In particular, RNA destined to be used as template for double-stranded cDNA synthesis in the generation of cDNA libraries, single-stranded cDNA synthesis for PCR or qPCR's, or for the synthesis of microarray target materials must be of the highest quality if researchers expect to obtain optimal results. RNA isolation techniques commonly employed for many other plant species are often insufficient in their ability to remove these contaminants from conifer samples and thus do not yield total RNA samples suitable for downstream manipulations. In this video we demonstrate methods for field collection of conifer tissues, beginning with the felling of a forty year-old tree, to the harvesting of phloem, secondary xylem, and reaction wood xylem. We also demonstrate an RNA isolation protocol that has consistently yielded high-quality RNA for subsequent enzymatic manipulations.

An Improved Method of RNA Isolation from Loblolly Pine P. taeda L. and Other Conifer Species

Many plant tissues, including phloem and xylem from loblolly pine (Pinus taeda L.), contain high levels of phenolics and polysaccharides that interfere with RNA purification. This presentation discusses techniques for the harvest of field-grown tissues and isolation of RNA of sufficient quality for microarrays and other genomic analyses.

Tissues isolated from conifer species, particularly those belonging to the Pinaceae family, such as loblolly pine (Pinus taeda L.), contain high ...

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 ...

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.

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 ...

Generation of Stable Human Cell Lines with Tetracycline-inducible (Tet-on) shRNA or cDNA Expression

A major approach in the field of mammalian cell biology is the manipulation of the expression of genes of interest in selected cell lines, with the aim to reveal one or several of the gene's function(s) using transient/stable overexpression or knockdown of the gene of interest. Unfortunately, for various cell biological investigations this approach is unsuitable when manipulations of gene expression result in cell growth/proliferation defects or unwanted cell differentiation. Therefore, researchers have adapted the Tetracycline repressor protein (TetR), taken from the E. coli tetracycline resistance operon1, to generate very efficient and tight regulatory systems to express cDNAs in mammalian cells2,3. In short, TetR has been modified to either (1) block initiation of transcription by binding to the Tet-operator (TO) in the promoter region upon addition of tetracycline (termed Tet-off system) or (2) bind to the TO in the absence of tetracycline (termed Tet-on system) (Figure 1). Given the inconvenience that the Tet-off system requires the continuous presence of tetracycline (which has a half-life of about 24 hr in tissue cell culture medium) the Tet-on system has been more extensively optimized, resulting in the development of very tight and efficient vector systems for cDNA expression as used here.
Shortly after establishment of RNA interference (RNAi) for gene knockdown in mammalian cells4, vectors expressing short-hairpin RNAs (shRNAs) were described that function very similar to siRNAs5-11. However, these shRNA-mediated knockdown approaches have the same limitation as conventional knockout strategies, since stable depletion is not feasible when gene targets are essential for cellular survival. To overcome this limitation, van de Wetering et al.12 modified the shRNA expression vector pSUPER5 by inserting a TO in the promoter region, which enabled them to generate stable cell lines with tetracycline-inducible depletion of their target genes of interest.
Here, we describe a method to efficiently generate stable human Tet-on cell lines that reliably drive either inducible overexpression or depletion of the gene of interest. Using this method, we have successfully generated Tet-on cell lines which significantly facilitated the analysis of the MST/hMOB/NDR cascade in centrosome13,14 and apoptosis signaling15,16. In this report, we describe our vectors of choice, in addition to describing the two consecutive manipulation steps that are necessary to efficiently generate human Tet-on cell lines (Figure 2). Moreover, besides outlining a protocol for the generation of human Tet-on cell lines, we will discuss critical aspects regarding the technical procedures and the characterization of Tet-on cells.

Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression

A rapid and simple way to generate human cell lines with inducible and reversible cDNA overexpression or shRNA-mediated knock-down of the gene of interest. This method enables researchers to reliably and highly reproducibly manipulate cell lines that are difficult to alter by transient transfection methods or conventional knockdown/knockout strategies.

A major approach in  the field of mammalian cell biology is the manipulation of the expression of  genes of interest in selected cell lines, with the aim ...

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes precursor mRNA beyond the end of mature RNAs, and an active process of endonuclease activity is required at a specific site. Cleavage of the precursor RNA normally occurs 10-30 nt downstream from the consensus polyA site (AAUAAA) after the CA dinucleotides. Proteins from the cleavage complex, a multifactorial protein complex of approximately 800 kDa, accomplish this specific nuclease activity. Specific RNA sequences upstream and downstream of the polyA site control the recruitment of the cleavage complex. Immediately after cleavage, pre-mRNAs are polyadenylated by the polyA polymerase (PAP) to produce mature stable RNA messages.
Processing of the 3&#8217; end of an RNA transcript may be studied using cellular nuclear extracts with specific radiolabeled RNA substrates. In sum, a long 32P-labeled uncleaved precursor RNA is incubated with nuclear extracts in vitro, and cleavage is assessed by gel electrophoresis and autoradiography. When proper cleavage occurs, a shorter 5&#8217; cleaved product is detected and quantified. Here, we describe the cleavage assay in detail using, as an example, the 3&#8217; end processing of HIV-1 mRNAs.

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

RNA polymerase II synthesizes a precursor RNA that extends beyond the 3' end of the mature mRNA. The end of the mature RNA is generated cotranscriptionally, at a site dictated by RNA sequences, via the endonuclease activity of the cleavage complex. Here, we detail the method to study cleavage reactions in vitro.