We would like to thank the following persons without whose help the collection of pine tissues would not have been possible: Dr. Joe Nairn, Matt Bryman, Michael Bordeaux, Ujwal Bagal, Huizhe Jin, and Amanda Bouffier.

Part 1: Tree Harvest
After the tree is selected and felled, it is important to work as carefully and as quickly as possible. As each different tissue type is harvested, they should be placed immediately into their own liquid nitrogen vessels to avoid any cross contamination of sample material. This RNA isolation protocol is scalable.
<ol> 
 <li>Cut bolts into lengths of approximately 0.5-0.75 m and with a chainsaw score the bark in two or three places following the longitudinal axis of the bolt.</li>
 <li>Using a wood chisel, work the edge of the scored region until the bark begins to separate from the wood and continue working the chisel while pulling back on the section of bark until it separates completely from the stem.</li>
 <li>Secondary xylem is harvested with a straight edge, single sided razor by scraping along the longitudinal axis of the bolt that has been stripped of bark. Wear latex gloves to minimize contamination of the tissue.</li>
 <li>Phloem, which is located on the inner side of the bark sections, is readily harvested by manual peeling.</li>
 <li>Reaction wood is collected from the underside of limbs after marking them with spray paint to indicate their original orientation with respect the ground.</li>
 <li>Limbs are cut into bolts of 1-2 m and scored on opposite sides along the longitudinal axis according to the approximate locations of the two types of reaction wood: opposite wood (on the top side of the limb) and compression wood (on the bottom side of the limb). The bark is separated from the limb as described above, except that only the bark covering each type of reaction wood is removed at one time. Secondary xylem or phloem is then collected as described above and placed in liquid nitrogen.</li>
 <li>Other samples, such as needles, shoot tips and young cones, can be harvested by hand or using pruning shears as required.</li>
 <li>Collected samples frozen in liquid nitrogen should be placed in marked bags or suitable containers and packed in dry ice for shipment back to the laboratory.</li>
 </ol>
Part 2: Freezer Mill Processing of Samples
 <ol>
 <li>Fill the Spex 6850 freezer mill reservoir with liquid nitrogen and place the impact bar into one of the grinding vials and fill with liquid nitrogen to pre-chill. Pour off liquid nitrogen and add sample to vial.</li>
 <li>Make sure that no excess liquid nitrogen remains in the milling chamber and that excess nitrogen gas is completely vented before the end cap is seated to seal the chamber. </li>
 <li>Insert the grinding vial into the carrier and close.</li>
 <li>Mill woody samples for two cycles of 1 minute/cycle at a rate setting of 14.</li>
 <li>Use a liquid nitrogen chilled spatula to remove the milled sample (wood flour) into a beaker that has been pre-chilled and contains a small amount of liquid nitrogen.</li>
 <li>Pour the liquid nitrogen/sample slurry into a storage container and place at -80&deg; C until needed. </li>
 </ol>
Part 3: RNA Isolation, Day 1
 <ol>
 <li> Place 20mL of RNA Isolation Buffer into a 50mL capped Falcon tube and add 400 &mu;L &beta;-mercaptoethanol, vortex briefly, then heat in a 60&deg; C water bath. </li>
 <li> Add 4 g of frozen tissue to each tube. Cap and shake vigorously by hand followed by vortexing for 10 seconds.</li>
 <li> Add an equal volume of chloroform to the tube and invert gently to mix. Place the tube on a rotating wheel and allow end-over-end mixing for 5 minutes.</li>
 <li> Decant the mixture into a 50 mL Oak Ridge tube and centrifuge at 12,000 (17,200 xg) rpm in a SS34 fixed-angle rotor for 5 minutes.</li>
 <li> Transfer the aqueous phase to a fresh Oak Ridge tube and add a half-volume of chloroform. </li>
 <li> Vortex for 1 minute and continue mixing on a rotating wheel for 5 minutes. Centrifuge at 12,000rpm (17,200 xg) in a SS34 fixed angle rotor for 5 minutes.</li>
 <li> Transfer the aqueous phase to fresh Oak Ridge tube and add a half-volume of chloroform. Vortex for 1 minute. Centrifuge at 12,000 rpm (17,200 xg) in a SS34 fixed angle rotor for 10 minutes.</li>
 <li> Transfer the aqueous phase to a 50 mL high speed polypropylene tube. Centrifuge at 10,000 rpm (11,900 xg) in a SS34 fixed angle rotor for 20 minutes.</li>
 <li> Decant supernatant into 50 mL Falcon tube and add a quarter-volume 10 M LiCl solution to supernatant. Briefly vortex and place at 4&deg; C for overnight precipitation.</li>
 </ol>
Part 4: RNA Isolation, Day 2
 <ol>
 <li> Heat SSTE buffer in a 65&deg; C water bath prior to starting.</li>
 <li> Pour the LiCl-precipitated sample into a polypropylene tube and pellet the RNA by centrifugation at 11,000rpm in a SS34 fixed angle rotor (14,450 xg) for 30 minutes at 4&deg; C.</li>
 <li> After centrifugation, pour off and discard the liquid, and invert the tubes on Kimwipes for approximately 1 minute. Pipette off any remaining supernatant. </li>
 <li> Resuspend each pellet in 800 &mu;L of heated SSTE using a pipette to thoroughly mix the samples. Transfer the resuspended sample to 2 mL Ambion RNAse-free microfuge tubes.</li>
 <li> Heat samples at 65&deg; C for 2 minutes followed by vigorous vortexing to ensure complete resuspension.</li>
 <li> Add an equal volume of phenol-chloroform, pH8, to each sample tube. Vortex each sample for 30 seconds and then spin in a microfuge at 14,000 rpm for 3 minutes</li>
 <li> Transfer the aqueous phase to a new 2mL Ambion RNAse-free microfuge tubes, and add an equal volume of chloroform. Vortex each sample for 30 seconds and spin in the microfuge at 14,000rpm for 3 minutes.</li>
 <li> Transfer aqueous phase to Phase-Lock Gel tubes that have been previously prepared by microfuging at 11,000 rpm for 2 minutes. Add a half-volume of chloroform and gently pipette to mix. </li>
 <li> Centrifuge at 11,000 rpm for 5 minutes and transfer the aqueous phase to fresh 2mL Ambion RNAse-free microfuge tubes.</li>
 </ol>
Part 5: RNA Precipitation and Ethanol Wash
 <ol>
 <li> Add 50 &mu;L 3.0 M sodium acetate, pH 4.8, and 1 mL 100% ethanol to the aqueous phase. Vortex and precipitate in -80&deg; C for 30 minutes or overnight at -20&deg; C.</li>
 <li> Microfuge samples at 14,000 rpm for 30 minutes at 4&deg; C, and carefully aspirate off the supernatant. Do not disturb the pellet.</li>
 <li> Wash the RNA pellets with 1 mL -20&deg; C 70% (v/v) ethanol. Microfuge at 14,000 rpm for 1 minute and aspirate off the ethanol. </li>
 <li> Repeat step 5.3.</li>
 <li> Uncap the tubes and air-dry pellets on the bench for 3-5 minutes.</li>
 </ol>
Part 6: Final Resuspension and Quantification
<ol>
	<li> Resuspend each pellet in 100 &mu;L DEPC-treated water. Mix by vortexing and incubate the tubes at 65&deg; C for 2 minutes before placing on ice. Repeat if necessary to ensure sample resuspension.</li>
 <li> Pool like samples if desired and make 50 &mu;L of a 1:10 dilution in 10mM Tris, pH 7.5, for spectrophotometric quantitation. Absorbance ratios for 260/280 and 260/230 are typically greater than 2.1 and 2.2, respectively. RNA isolation yields range from 60-120&mu;g/g frozen tissue.</li>
 <li> Analyze samples by running 1.5-2.5 &mu;g RNA on a 1% agarose gel in TAE buffer. For more critical samples, analyze the RNA using an Agilent 2100 Bioanalyzer as per the manufacturer's instructions.</li>
 </ol>
Representative Results:
 RNA isolation yields from different conifer tissues range from 60-120 &mu;g/g frozen tissue with woody samples typically yielding around 70-80g/g frozen tissue. Diagnostic ratios of 260/280 and 260/230 are both typically greater than 2.1, and are good indicators that the RNA sample is free of any significant phenolic or carbohydrate contamination, respectively. Samples analyzed by agarose gel electrophoresis followed by EtBr staining should show three distinct bands for 25S, 18S, and 5S RNA (Figure 1). Determining the stoichiometry of the 28S to 18S on agarose gels is somewhat subjective, and factors such as running conditions for the gel, staining, and sample load can all have an effect. In general, the 28S:18S ratio will appear to be &gt; 1.5. However, some conifer samples have lower ratios. For example, following Agilent 2100 analysis we have seen several instances of conifer samples from different tissue types where the stoichiometry is closer to 1.2:1. Thus, an apparent low 28S:18S stoichiometry by agarose gel electrophoresis does not necessarily indicate a poor-quality sample. Leaf, shoot, and cone samples will often have additional distinct bands present due to chloroplastic ribosomal RNAs. EtBr-stained materials that do not migrate from the sample well or high molecular mass bands (&gt;8-10 kb) usually indicate genomic DNA contamination (Figure 2). Low molecular mass smears in the vicinity of or below the 5S band and reduced ribosomal band staining or an apparent 18S &gt; 28S stoichiometry is a good indicator that RNA degradation has occurred (Figure 3). In the Agilent 2100 electropherogram analysis, the base line should be low and relatively smooth with major peaks corresponding to 18S and 28S ribosomal RNAs having 28S:18S ratios ranging anywhere from 1.2 to 2.0. The Agilent RNA Integrity Number (RIN) value may be a better predictor of RNA quality and should be at least 7 or greater for RNA that is to be used for the preparation of microarray targets. Figure 4 shows a typical Agilent 2100 electropherogram for xylem RNA with a 28S:18S ratio equal to 1.4 and a RIN value of 8.6 while Figure 5 shows a poor xylem RNA prep having a very high baseline and noticeable degradation as revealed by the 28S:18S ratio equal to 0.65 and a RIN value of 3.4.

<img src="/files/ftp_upload/1751/1751fig1.jpg" alt="Figure 1" /> Figure 1. Agarose gel analysis of high-quality pine RNA. Lane 1, NEB 1 kb standard, Lane 2 shows a typical total RNA isolation from loblolly pine secondary xylem with characteristic ribosomal 28S, 18S, and 5S bands and apparent 28S:18S ratio &gt;1.
<img src="/files/ftp_upload/1751/1751fig2.jpg" alt="Figure 2" /> Figure 2.Agarose gel analysis of pine RNA sample contaminated with genomic DNA. Lane 1, NEB 1 kb standard, Lane 2, xylem RNA sample showing genomic DNA contamination.
<img src="/files/ftp_upload/1751/1751fig3.jpg" alt="Figure 3" /> Figure 3.Agarose gel analysis of pine sample showing RNA degradation and contamination with genomic DNA. Lane 1, NEB 1 kb standard, Lane2, xylem RNA sample showing genomic DNA contamination and severe RNA degradation.
<img src="/files/ftp_upload/1751/1751fig4.jpg" alt="Figure 4" /> Figure 4.Agilent 2100 electropherogram of xylem total RNA isolated using the described method on loblolly pine xylem. 28S:18S ratio equals 1.4, RIN value equals 8.6 
<img src="/files/ftp_upload/1751/1751fig5.jpg" alt="Figure 5" /> Figure 5.Agilent 2100 electropherogram of apical tip total RNA showing severe degradation and genomic contamination. 28S:18S ratio equals 0.65, RIN value equals 3.4

<ol>
	<li>Chang, S., Puryear, J., Cairney, 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+simple+and+efficient+method+for+extracting+RNA+from+pine+trees.">A simple and efficient method for extracting RNA from pine trees.</a> Plant Molecular Biology Reporter. 11 (2), 113-116 (1993).</li><li>Lorenz, W. W., Yu, Y. S., Sim&#369;es, M., 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=Processing+the+Loblolly+Pine+PtGen2+cDNA+Microarray.">Processing the Loblolly Pine PtGen2 cDNA Microarray.</a> Journal of Visualized Experiments. 25, (2009).</li></ol>

Obtaining high-quality RNA from conifer species can be a difficult task given the high levels of phenolic and polysaccharide compounds found in woody tissues. Starting with the protocol developed by Chang et al. (1), we have found that more rigorous extraction and clean up steps lead to the consistent isolation of very high-quality total RNA from various woody and non-woody tissues sampled from a wide variety of conifer species. This video has demonstrated not only our modified RNA isolation protocol, but also the step...

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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>RNA Isolation Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>2% CTAB (hexadecyltrimethylammonium bromide), 2% PVP (polyvinyl pyrrolidinone; Mw 30-40,000), 100mM Tris-HCl (pH8.0), 25mM EDTA, 2.0M NaCl, 0.5g/L Spermidine</td>
</tr>
<tr>
<td>Chloroform</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>C298-4</td>
<td>&nbsp;</td>
<tr>
<td>Phenol, Ultrapure</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15509-037</td>
<td>&nbsp;</td>
<tr>
<td>10M LiCl</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Made with DEPC-treated water</td>
</tr>
<tr>
<td>SSTE Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1M NaCl, 0.5% SDS, 10mM Tris-HCl (pH8.0), 1mM EDTA (pH8.0)</td>
</tr>
<tr>
<td>Sodium acetate (NaOAc) 3M pH4.8</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Made with DEPC-treated water</td>
</tr>
<tr>
<td>Phenol-chloroform (pH8.0)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>120mL phenol, 160mL chloroform titrated with multiple changes of 0.5M Tris-Cl pH 8.0</td>
</tr>
<tr>
<td>Oak Ridge High Speed Teflon Tubes</td>
<td>&nbsp;</td>
<td>Thermo-Fisher</td>
<td>#05-562-16B</td>
<td>&nbsp;</td>
<tr>
<td>Polypropylene 50mL High Speed Tubes</td>
<td>&nbsp;</td>
<td>Thermo-Fisher</td>
<td>#05-562-10K</td>
<td>&nbsp;</td>
<tr>
<td>BD Falcon,sterile, capped 50mL conical disposable tubes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>#21008-939</td>
<td>&nbsp;</td>
<tr>
<td>Phase Lock Gel Heavy, 2mL</td>
<td>&nbsp;</td>
<td>Thermo-Fisher</td>
<td>#2302830</td>
<td>&nbsp;</td>
<tr>
<td>Ambion RNase-free Microfuge Tubes, 2mL</td>
<td>&nbsp;</td>
<td>Ambion, Inc.</td>
<td>#AM12425</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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.

Watch this Scientific Journal Video about An Improved Method of RNA Isolation from Loblolly Pine P. taeda L. and Other Conifer Species at JoVE.com

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.

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 ...

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Research

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13.8K vistas.  University of Georgia (UGA). Muchos tejidos de plantas, incluyendo el floema y el xilema de pino taeda ( Pinus taeda L.), contienen altos niveles de compuestos fenólicos y polisacáridos que interfieren con la purificación de ARN. Esta presentación se analizan las técnicas para la cosecha de tejidos cultivadas en el campo y el aislamiento de ARN de calidad suficiente para microarrays y otros análisis genómico.

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

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 ...

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 ...

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The model organism C. elegans uses pseudocoelomic fluid as a passive circulatory system. Direct assay of this fluid has not been previously possible. Here we present a novel technique to directly assay the extracellular space, and use systemic silencing signals during an RNAi response as a proof of principle example.

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ...

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a &#34;drop tube&#34; to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Stomata Tape-Peel: An Improved Method for Guard Cell Sample Preparation

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is often difficult to isolate and thus studying them often provides a challenge. 
This study establishes a stomatal guard cell enrichment method. The protocol utilizes Scotch tape to isolate guard cells and extract proteins from the cells. This method improves the integrity and yield of guard cell during isolation and provides a reliable sample for the study of cell signaling processes and how they correlate to stomatal movement phenotypes. In this method, the plant leaves were partitioned between two portions of tape and peeled apart to remove the abaxial side. This protocol was applied to Arabidopsis leaf tissue to isolate guard cells. The cells were treated with fluorescein diacetate (FDA) to determine viability and with abscisic acid (ABA) to assess stomata movement in response to ABA. In conclusion, this protocol proves useful for preparing enriched stomatal guard cells and isolating proteins. The quality of the cells and proteins obtained here enable physiological and other biological studies.

This protocol describes a method of preparing enriched stomatal guard cells that is useful for physiological and other biological studies.

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is ...

An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on different needs, like time, labor, cost, and primary hepatocyte usage, resulting in various primary hepatocyte isolation protocols. However, the numerous steps and time-consuming reagent preparations in primary hepatocyte isolation are major drawbacks for efficiency. After comparing different protocols for their pros and cons, the advantages of each were combined, and a rapid and efficient primary hepatocyte isolation protocol was formulated. Within only ~35 min, this protocol could yield as much, if not more, healthy primary hepatocytes as other protocols. Further, glucose metabolism experiments performed using the isolated primary hepatocytes validated the usefulness of this protocol in in vitro liver metabolism studies. We also extensively reviewed and analyzed the significance and purpose of each step in this study so that future researchers can further optimize this protocol based on needs.

Primary hepatocytes are a valuable tool to study liver response and metabolism in vitro. Utilizing commercially available reagents, an improved time- and labor-efficient protocol for mouse primary hepatocyte isolation was developed.

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on ...