Name: high-throughput, robust and highly time-flexible method for surface sterilization of arabidopsis seeds
Uploaded: 2021-10-04T14:15:00
Description: Watch this Scientific Journal Video about High-throughput, Robust and Highly Time-flexible Method for Surface Sterilization of Arabidopsis Seeds at JoVE.com

This research was funded by the Autonomous Province of Trento through core funding of the Ecogenomics group of Fondazione E. Mach.

1. Reagents and media preparation
<ol>
	<li>Prepare 70% ethanol solution: Add 737 mL of 95% technical ethanol to 263 mL of distilled water. Mix thoroughly. 
	NOTE: Prepare 70% ethanol solution on a non-sterile working bench. 
	CAUTION: Ethanol is highly flammable and can cause serious irritation to the eyes. Keep away from flames and heat sources. In case of contact with eyes, rinse with abundant water.</li>
	<li>Prepare 5% bleach solution: Add 5 mL of household bleach (containing ~3.5% of Sodium hypochlorite...

<ol>
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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drought+and+salt+stress+tolerance+of+an+arabidopsis+glutathione+S-transferase+U17+knock-out+mutant+are+attributed+to+the+combined+effect+of+glutathione+and+abscisic+acid.">Drought and salt stress tolerance of an arabidopsis glutathione S-transferase U17 knock-out mutant are attributed to the combined effect of glutathione and abscisic acid.</a> Plant Physiology. 158 (1), 340-351 (2012).</li><li>Li, D. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+a+large+dataset+indicates+that+internal+transcribed+spacer+(ITS)+should+be+incorporated+into+the+core+barcode+for+seed+plants.">Comparative analysis of a large dataset indicates that internal transcribed spacer (ITS) should be incorporated into the core barcode for seed plants.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (49), 19641-19646 (2011).</li><li>Mathur, J., Koncz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+maintenance+of+cell+suspension+cultures.+Arabidopsis+Protocols.">Establishment and maintenance of cell suspension cultures. Arabidopsis Protocols.</a> Methods in Molecular Biology. 82, 27-30 (1998).</li><li>Li, M., Cappellin, L., Xu, J., Biasioli, F., Varotto, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+for+in+planta+characterization+of+VOC+biosynthetic+genes+by+PTR-ToF-MS.">High-throughput screening for in planta characterization of VOC biosynthetic genes by PTR-ToF-MS.</a> Journal of Plant Research. 133 (1), 123-131 (2020).</li><li>Li, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+planta+recapitulation+of+isoprene+synthase+evolution+from+ocimene+synthases.">In planta recapitulation of isoprene synthase evolution from ocimene synthases.</a> Molecular Biology and Evolution. 34 (10), 2583-2599 (2017).</li><li>Li, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+isoprene+emission+in+Arecaceae+(palms).">Evolution of isoprene emission in Arecaceae (palms).</a> Evolutionary Applications. 14, 902-914 (2020).</li><li>Murashige, T., Skoog, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+revised+medium+for+rapid+growth+and+bio+assays+with+tobacco+tissue+cultures.">A revised medium for rapid growth and bio assays with tobacco tissue cultures.</a> Physiologia Plantarum. 15 (3), 473-497 (1962).</li><li>Bent, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+thaliana+floral+dip+transformation+method.">Arabidopsis thaliana floral dip transformation method.</a> Methods in Molecular Biology. 343, 87-104 (2006).</li><li>Lundberg, D. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+arsenic+stress+response+by+ethylene+biosynthesis+and+signaling+in+Arabidopsis+thaliana.">Regulation of arsenic stress response by ethylene biosynthesis and signaling in Arabidopsis thaliana.</a> Environmental and Experimental Botany. 185, 104408 (2021).</li><li>Lindsey, B. E., Rivero, L., Calhoun, C. S., Grotewold, E., Brkljacic, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+method+for+high-throughput+sterilization+of+Arabidopsis+seeds.">Standardized method for high-throughput sterilization of Arabidopsis seeds.</a> Journal of Visualized Experiments: JOVE. 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Sterilization of seeds is the fundamental step for functional studies in Arabidopsis. Although it is frequently carried out for many different purposes, limited studies on high-throughput seed surface sterilization in Arabidopsis are available.
So far, one of the methods with the highest throughput is using chlorine gas generated by mixing bleach with concentrated HCl. Although this method requires limited hands-on time, it uses a gas highly toxic to human beings27. In ...

Arabidopsis is a diploid plant species belonging to the Brassicaceae family. Its relatively short life cycle (two months per generation under long-day growing conditions), small plant size, and self-pollination with the production of hundreds of seeds per plant have made it the first fundamental plant model species1,2. In addition, its genome was fully sequenced3, extensive reverse genetics tools (saturated T-DNA, transposon, and chemically mutagenized populations) are available4,5,6, and effective Agrobacterium-mediated transformation is well-established to obtain sufficient transgenic lines for further downstream work7. Thus, during the last two decades, great advances have been achieved using Arabidopsis as a model species to dissecting diverse aspects of plant biology at the molecular level, including natural, genetic and phenotypic variation8,9.
To functionally characterize genes of interest in Arabidopsis, seed surface sterilization to eliminate fungal and bacterial contaminants is the prerequisite step for many downstream protocols requiring axenic cultures. Genetic transformation for the overexpression10, knock-down (RNA-I11) or knock-out (genome editing12,13) of gene function, subcellular localization14, promoter activity15,16, protein-protein17 and protein-DNA interaction18, to cite only the most common applications, all necessitate a seed surface sterilization step. Thus, despite its relative simplicity, seed surface sterilization plays a fundamental role in many functional analyses.
So far, two major categories of seed surface sterilization methods have been developed based either on gas- or on liquid-phase sterilization19. While the throughput of gas-phase seed surface sterilization is medium to high, using the hazardous reagent chlorine gas as a surface sterilization agent has hindered its wide application. Methods based on liquid-phase sterilization, on the contrary, rely on milder chemicals like ethanol and bleach solutions for surface sterilization, and they are more widely used despite they have an inherently lower throughput than chlorine fumigation. In general, two different methods which use liquid reagents are commonly used. One largely used method is based on washing with ethanol and bleach at different concentrations for different duration of time20,21. Another method is based on the application of bleach only21,22. Both methods are mainly applied for small-scale seed surface sterilization. However, in many experiments, it is necessary to screen many Arabidopsis transgenic lines derived from one transformation15,23 or screen in parallel many transgenic lines generated from different transformations24,25. To the best of our knowledge, no liquid-based method for high-throughput seed surface sterilization has been published, which constitutes, although little-recognized, an important bottleneck for functional genomics approaches. Therefore, developing safe, robust, and high-throughput methods for seed surface sterilization is a necessary and critical step towards the success of the functional characterization of many genes at once.
To this end, in the current study, an improved method for surface sterilization of Arabidopsis seeds is presented. This method is safe, low cost, highly robust, and high-throughput, allowing handling 96 independent lines within one hour from the beginning of seed surface sterilization until the end of seed sowing in Petri dishes. The method demonstrated relies on widely available, basic laboratory instrumentation like a vacuum pump, consumable glassware, and plastic ware. This improved method provides the scientific community a safe, simple, and affordable approach to streamline seed surface sterilization with a throughput adequate to modern functional genomics approaches in Arabidopsis and other non-model plant species.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aquarium valve</td>
			<td>Amazon</td>
			<td>B074CYC5SD</td>
			<td>Kit including 2 valves and thin-walled tubings. The valve prevents the liquids to go back to the sterile tip</td>
		</tr>
		<tr>
			<td>Arabidopsis Col-0 wild-type seeds</td>
			<td>Nottingham Arabidopsis Stock Center</td>
			<td>N1093</td>
			<td>Wild type seeds (sensitive to kanamycin)</td>
		</tr>
		<tr>
			<td>Arabidopsis transgenic line AdoIspS-79 seeds</td>
			<td>NA</td>
			<td>NA</td>
			<td>Transgenic line overexpressing an isoprene synthase gene from Arundo donax transformed in the Col-0 background, resistant to kanamycin (Li et al. (2017) Mol. Biol. Evol., 34, 2583&#8211;2599). Available on request from the authors</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Eppendorf</td>
			<td>EP022628188</td>
			<td>Benchtop microcentrifuge used for spinning down the seeds</td>
		</tr>
		<tr>
			<td>Murashige &#38; Skoog medium including vitamins</td>
			<td>Duchefa</td>
			<td>M0222</td>
			<td>Standard medium for plant sterile culture</td>
		</tr>
		<tr>
			<td>Pipette controller</td>
			<td>Brand</td>
			<td>26300</td>
			<td>Used to operate the serological pipette</td>
		</tr>
		<tr>
			<td>Polyethylene tube 1</td>
			<td>Roth</td>
			<td>9591.1</td>
			<td>Tube for connection from vacuum pump to decantation bottle (inner diameter: 7 mm; outer diameter: 9 mm)</td>
		</tr>
		<tr>
			<td>Polyethylene tube 2</td>
			<td>Roth</td>
			<td>9587.1</td>
			<td>Tube for connection from decantation bottle to the aquarium valve&#160; (inner diameter: 5 mm; outer diameter: 7 mm)</td>
		</tr>
		<tr>
			<td>Screw cap with connectors</td>
			<td>Roth</td>
			<td>PY86.1</td>
			<td>2-way dispenser screw cap GL45 in polypropylene for decanting bottle</td>
		</tr>
		<tr>
			<td>Serological pipette</td>
			<td>Brand</td>
			<td>27823</td>
			<td>Graduated glass (reusable) serological pipette. Disposable pipettes can be used instead</td>
		</tr>
		<tr>
			<td>Shakeret al.</td>
			<td>Qiagen</td>
			<td>85300</td>
			<td>TissueLyser II bead mill used normally for tissue homogenization. Without the addition of beads to the tubes it works as shaker.</td>
		</tr>
		<tr>
			<td>Technical ethanol</td>
			<td>ITW Reagents (Nova Chimica Srl)</td>
			<td>212800</td>
			<td>Ethanol 96% v/v partially denatured technical grade</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Merck Millipore</td>
			<td>655205</td>
			<td>Non-ionic detergent acting as surfactant</td>
		</tr>
		<tr>
			<td>Universal tubing connectors</td>
			<td>Roth</td>
			<td>Y523.1</td>
			<td>Can be used to improve/simplify tubing connections</td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>Merck Millipore</td>
			<td>WP6222050</td>
			<td>Used for making the suction device</td>
		</tr>
	</tbody>
</table>

high-throughput, robust and highly time-flexible method for surface sterilization of arabidopsis seeds

Arabidopsis is by far the plant model species most widely used for functional studies. The surface sterilization of Arabidopsis seeds is a fundamental step required towards this end. Thus, it is paramount to establish high-throughput Arabidopsis seed surface sterilization methods to handle tens to hundreds of samples (e.g., transgenic lines, ecotypes, or mutants) at once. A seed surface sterilization method based on the efficient elimination of liquid in tubes with a homemade suction device constructed from a common vacuum pump is presented in this study. By dramatically reducing labor-intensive hands-on time with this method handling several hundreds of samples in one day is possible with little effort. Series time-course analyses further indicated a highly flexible time range of surface sterilization by maintaining high germination rates. This method could be easily adapted for surface sterilization of other kinds of small seeds with simple customization of the suction device according to the seed size, and the speed desired to eliminate the liquid.

In order to assess the time required for the entire seed sterilization procedure, the time differences for liquid handling 96 samples in the current protocol were calculated and compared with traditional pipetting methods. The result indicates that the current protocol saves time, cutting the liquid handling time to one-fourth of that with the traditional protocols (Table 1). The table further highlights that the liquid removal time in the current protocol saves more time than that of the traditional met...

Watch this Scientific Journal Video about High-throughput, Robust and Highly Time-flexible Method for Surface Sterilization of Arabidopsis Seeds at JoVE.com

High-throughput, Robust and Highly Time-flexible Method for Surface Sterilization of Arabidopsis Seeds

A high-throughput protocol for the surface sterilization of Arabidopsisthaliana (Arabidopsis) seeds is provided, optimizing the liquid handling steps with a simple suction device constructed with a vacuum pump. Hundreds of seed samples can be surface-sterilized in one day.

Arabidopsis is by far the plant model species most widely used for functional studies. The surface sterilization of Arabidopsis seeds is a fundamental ...

This protocol improve seed surface sterilization, a fundamental step in functional genomics of the model species Arabidopsisthaliana. The main advantages of this technique are ease and high throughput, allowing the processing of hundreds of samples per day. With some simple modifications, this method can be applied to many other model and non-model plant species.For first-time users, please pay attention to the temperature of the medium and the centrifugation speed as they are both critical for seed survival. To begin, prepare 70%ethanol by adding 95%technical ethanol to distilled water and mixing thoroughly. Next, prepare 5%bleach solution by adding five milliliters of household bleach to 95 milliliters of sterile distilled water.Then, add a few drops of nonionic detergent to the bleach solution, and mix thoroughly. Next, prepare half-strength Murashige and Skoog medium by adding 2.2 grams of MS medium powder including vitamins and 10 grams of sucrose in 800 milliliters of distilled water. Adjust the pH of the medium to 5.7 using one molar potassium hydroxide, then bring the volume up to one liter using distilled water.Aliquot 500 milliliters of the medium into a one-liter bottle, and add four grams of agar to prepare a solid medium. After autoclaving, allow the medium to cool down to 50 to 53 degrees Celsius in a water bath. Then pour it into Petri dishes under the laminar flow hood.To prepare selective medium, add kanamycin to the medium, and pour it into Petri dishes as demonstrated earlier. To set up the aspirator, connect the vacuum pump inlet to one end of a polyethylene tube of a suitable size. Then connect the other end of the tube to the outlet of the two-way lid of the decantation bottle.Wrap the tubing's junction tightly with a sealing film to ensure airtight connection. Next, connect a second polyethylene tube to the inlet of the screw cap on the decantation bottle. Then connect the other side of the tube to the outlet of an aquarium valve fitted with a thin polyethylene tube.If necessary, wrap the junction with the sealing film to eliminate air leakage. After labeling two batches of 48 1.5-milliliter microcentrifuge tubes with progressive numbers, add 100 to 200 Arabidopsis seeds to each of the 96 sterile microcentrifuge tubes, roughly one to two millimeters above the bottom of the conical end of the tube. Next, using a 10-milliliter sterile serological pipette, add around one milliliter of 70%ethanol into each tube, and carefully close the lids.Shake the tubes at an oscillation frequency of eight hertz for three minutes in a shaker. Then remove the adapters from the shaker and transfer them into the basket of a benchtop microcentrifuge for spinning down the seeds using the pulse function. After transferring the tubes to a rack, open all tubes under the laminar flow hood.Do not touch the part of the lids fitting into the tubes to avoid contamination. Then, under the laminar flow hood, fit a sterile 200-microliter yellow tip onto the aquarium valve inlet of the homemade aspirator, and switch on the pump. Insert the tip just above the level of the seeds to avoid touching the seeds when sucking the liquid.Next, using a 10-milliliter sterile serological pipette, aliquot one milliliter of 5%bleach solution into each tube. Close the lids before placing the tubes back into the shaker adapters, then shake the tubes as demonstrated previously. After spinning down the seeds using the pulse function of a benchtop centrifuge, fit a new sterile 200-microliter yellow tip onto the aquarium valve, and switch on the pump.Insert the tip above the level of the seeds to avoid touching the seeds when sucking the bleach solution. Next, using a 10-milliliter sterile serological pipette, aliquot one milliliter of sterilized water into each tube. After fitting a new sterile 200-microliter yellow tip onto the aquarium valve, switch on the pump.Insert the tip just above the level of the seeds to avoid touching the seeds when sucking the water. Finally, aliquot 500 microliters of sterilized water into each tube, and close all the lids in the laminar flow hood. The seeds are now ready to be sown.If required, keep the tubes at room temperature for up to a few hours or at four degrees Celsius overnight. Using a one-milliliter pipette, transfer the seeds with 300 to 400 microliters of sterile water into a Petri dish. After transferring 10 tubes, pour 1.5 to two milliliters of melted half-strength Murashige and Skoog medium without antibiotics into each plate.Quickly swirl the plate to distribute the seeds, and tape the plates on opposite sides. Wrap the plates in plastic or aluminum foil, and place them in a refrigerator for three days in the dark to obtain uniform germination. Germination analyses performed at days two, three, four, and seven indicated no significant differences among 10 to 40 minutes of sterilization time with 70%ethanol.However, when the sterilization time exceeded 40 minutes, the germination rates declined. Correspondingly, green cotyledon emergence rates also decreased. Based on the study, three minutes for 70%ethanol and three minutes for 5%bleach were selected as the minimum time to sterilize the seeds.To demonstrate that the seeds used originally were contaminated with microorganisms, non-sterile seeds were sown directly on the plates. Compared to sterile seeds, non-sterile seeds showed a fungal appearance after two days, which spread all over the plates after a seven-day germination. A cross-contamination assay performed using a single sterile pipette tip to process different seed samples showed that no green cotyledons were observed in the Columbia-0 genotype seeds sown in plates with kanamycin.In parallel, in Columbia-0 seeds sown in plates without kanamycin, all cotyledons appeared green after germination. These results indicate no carryover of contamination between samples despite using a single pipette tip to remove the sterile solution. This procedure is the basis for many other functional genomics methods like gene overexpression, genome editing, subcellular localization, promoter activity, and protein-protein and protein-DNA interactions.

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Research

JoVE Journal

Biology

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide resolution. This technique captures miRNAs by attaching 3&#8217; and 5&#8217; oligonucleotide adapters to miRNA molecules and allows de novo miRNA discovery. Coupling with powerful next-generation sequencing platforms, miR-Seq has been instrumental in the study of miRNA biology. However, significant biases introduced by oligonucleotide ligation steps have prevented miR-Seq from being employed as an accurate quantitation tool. Previous studies demonstrate that biases in current miR-Seq methods often lead to inaccurate miRNA quantification with errors up to 1,000-fold for some miRNAs1,2. To resolve these biases imparted by RNA ligation, we have developed a small RNA ligation method that results in ligation efficiencies of over 95% for both 3&#8217; and 5&#8242; ligation steps. Benchmarking this improved library construction method using equimolar or differentially mixed synthetic miRNAs, consistently yields reads numbers with less than two-fold deviation from the expected value. Furthermore, this high-efficiency miR-Seq method permits accurate genome-wide miRNA profiling from in vivo total RNA samples2.

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.