The authors would like to thank A Viks&oslash;-Nielsen (Novozymes) for the gift of the cellulolytic enzymes. This work was funded by FP7 RENEWAL and by BBSRC projects BB/G016178 and BB/G016194.

1. Preparation of the samples
We normally work with stems from either herbaceous or woody plants. In the case of herbaceous materials we grow the plants to maturity, and after seed development we collect dry stems once senescence is complete. The stems are chopped into 4 mm segments and placed in 2 ml vials with three milling balls. In the case of woody materials, the samples are ground to coarse sawdust with a course wood file and then placed in a ball mill for further processing.
The samples are placed in a rack within the grinding/formatting station (Figure 1). This station sequentially grinds, mixes, and weighs each sample. The number of repetitions needed per sample is established by testing the ground material in a series of preliminary experiments where the intrinsic variability for a certain particle size is determined. The 2 mL vials are pierced at the bottom and the materials are dispensed by vibration of the vial over the selected well. The vibrating arm is controlled by a feedback from the balance allowing for the accurate dispensing of the powder. The robot dispenses 4 mg/well in a standard analysis and 4 repetitions are needed in most the cases. This allows 20 plant samples/plate to be analysed.
2. Pretreatment
Once the samples are formatted, the 96-well plates are processed by an automated liquid handling system (Figure 2). Here a mild pretreatment is performed on the plant materials by adding 350 &mu;L of acid or alkaline solutions and heating the plate on an adapted block. To avoid evaporation during the analysis, the 96 well plates are sealed with a silicone matt. The temperature and time of the pretreatment can be modified according to the materials being studied. The maximum temperature, however, is 100 &deg;C. An alternative procedure for testing harsher pretreatments is to pretreat the materials off line and eliminate the pretreatmet from the process.
3. Hydrolysis
<ol>
 <li>After the materials are pretreated, the robot automatically removes the pretreatment solution, and washes, all the wells, 5 times with 850 &mu;L 25 mM Acetate Buffer. The rinses are carried out by adding buffer to the pretreatment solution, and subsequently removing 50% of the total volume after allowing the solids in the sample to settle. The height of the aspiration is set at half of the liquid mass to avoid aspirating solids from the bottom of the well; there is negligible loss of solids using this approach. After these washes the pH in the well is 4.5 and the material is ready for enzymatic hydrolysis.</li>
 <li>An enzyme mixture containing a solution of cellulases and hemicellulases is added by the robot. Into each well, 850 &mu;L of enzyme mixture is dispensed and the plate is moved into a 50 &deg;C shaking incubator. The standard hydrolysis is performed for 8 h, but this time can be adapted to the purpose of the experiment. The standard enzyme loading used for grasses is 7 FPU/g of material.</li>
</ol>
4. Detection of reducing sugars
Determination of reducing sugars released after hydrolysis was performed using a modified 3-methyl-2-benzothiazolinone hydrozone (MTBH) method3. MBTH was selected as the most suitable method because it was the easiest to automate and the least susceptible to interference from compounds such as proteins. We modified this highly sensitive method for use on the robotic platform so that it could accurately quantify sugars at the concentrations present in the biomass hydrolysates, with a final volume of 250 &mu;L which is suitable for a standard optical 96 well plate. All the steps below are performed automatically and three independent determinations of the reducing sugars were carried out for each saccharification reaction.
<ol>
 <li>30 &mu;L of hydrolyzate was taken from the deep well plate and mixed with 45 &mu;L of 25 mM Sodium Acetate buffer in a skirted PCR 96 well plate.</li>
 <li>25 &mu;L of 1N NaOH and 50 &mu;l of a solution containing 0.43 mg/ml MBTH and 0.14 mg/ml DTT were added.</li>
 <li>After mixing, the PCR plate was heated at 60&deg;C for 20 min in a thermocycler or similar device that delivers exactly the same amount of heat to all 96 wells.</li>
 <li>The resulting reaction was transferred to an optical plate.</li>
 <li>Add 100 &mu;l of oxidising reagent (0.2% FeNH4(SO4)2, 0.2% sulfamic acid and 0.1% HCl). Mix well and leave to develop for at least 1 h.</li>
 <li>Each plate should also contain standard reactions of 50 nmol, 100 nmol and 150 nmol glucose. The optical plates are read at 620 nm.</li>
</ol>
5. Representative Results: 
Examples of different types of analyses are presented in figure 3 and 4. All the results were obtained by using the automated platform. Figure 3 represents the increase in reducing sugars released by 8 h incubations from ground poplar with increasing amounts of cellulolytic enzymes. Figure 4 presents the effects of acid and alkaline pretreatment on poplar samples. The NaOH pretreatment is more efficient that the diluted H2SO4 pretreatment in facilitating the release of sugars during hydrolysis. The amount of sugars measured after hydrolysis decreases when the pretreatment is performed using NaOH concentrations higher than 1M.
<img src="/files/ftp_upload/3240/3240fig1.jpg" alt="Figure 1" /> 
Figure 1. Robotic station for grinding and 96 well formatting of biomass
<img src="/files/ftp_upload/3240/3240fig2.jpg" alt="Figure 2" /> 
Figure 2. Liquid handling station for the High Throughput saccharification analysis
<img src="/files/ftp_upload/3240/3240fig3.jpg" alt="Figure 3" /> 
Figure 3. Effect of different enzyme loadings on the release of reducing sugar equivalents from poplar samples
<img src="/files/ftp_upload/3240/3240fig4.jpg" alt="Figure 4" /> 
Figure 4. Effect of different pretreatments on the saccharification of poplar samples. A. Sugars released after pretreament with different percentages of H2SO4 at 90&deg;C for 30 minutes. B. Sugars released after NaOH pretreatments at the same temperature and time than the acid pretreatment.

<ol>
	<li>Carpita, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+Biogenesis+of+the+Cell+Walls+of+Grasses.">Structure and Biogenesis of the Cell Walls of Grasses.</a> Annu Rev Plant Physiol Plant Mol Biol. 47, 445-476 (1996).</li><li>Gomez, L. D., Steele-King, C. G., McQueen-Mason, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustainable+liquid+biofuels+from+biomass:+the+writing's+on+the+walls.">Sustainable liquid biofuels from biomass: the writing's on the walls.</a> New Phytol. 178, 473-485 (2008).</li><li>Anthon, G. E., Barrett, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+reducing+sugars+with+3-methyl-2-benzothiazolinonehydrazone.">Determination of reducing sugars with 3-methyl-2-benzothiazolinonehydrazone.</a> Anal Biochem. 305, 287-289 (2002).</li><li>Gomez, L. D., Whitehead, C., Barakate, A., Halpin, C., McQueen-Mason, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+saccharification+assay+for+determination+of+digestibility+in+plant+materials.">Automated saccharification assay for determination of digestibility in plant materials.</a> Biotechnol Biofuels. 3, 23-23 (2010).</li></ol>

We have nothing to disclose.

Variations of the standard saccharification protocol can be used in the same platform for determining the activity of cellulolytic enzymes (i.e. by variation of the enzyme concentrations used in a plate as well as using paper as substrate); comparison of the efficiency of several enzyme mixtures on a specific material; time course for saccharification; etc.
A standard saccharification protocol to compare the saccharification potential in different plant materials involves an eight hour hydrolysis (Figures 3 and 4). Most of the plant materials analysed requires four replicates. Under these conditions, the platform can process 80 samples/day. This analysis is being used to screen large populations of barley, maize, and brachypodium in order to establish variability in saccharification potential and the genes involved in its determination4.

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
		</tr>
		<tr>
			<td>Grinding &amp; weighing robot</td>
			<td>Labman Automation</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2 ml micro tube with caps</td>
			<td>Sarstedt Ltd</td>
			<td>72.694</td>
		</tr>
		<tr>
			<td>5 mm stainless steel beads</td>
			<td>Qiagen Ltd</td>
			<td>69989</td>
		</tr>
		<tr>
			<td>1.2ml Abgene square well storage plates</td>
			<td>Fisher Scientific Ltd</td>
			<td>TUL-050-050C</td>
		</tr>
		<tr>
			<td>Whatman cap mat for 96 square well plates </td>
			<td>Fisher Scientific Ltd</td>
			<td>7704-0104</td>
		</tr>
		<tr>
			<td>Liquid hanling robot</td>
			<td>Tecan Group Ltd.</td>
			<td>Freedom Evo 200</td>
		</tr>
		<tr>
			<td>Sulphuric acid </td>
			<td>Fisher Scientific Ltd</td>
			<td>S/9231/PB17</td>
		</tr>
		<tr>
			<td>Sodium hydroxide </td>
			<td>Fisher Scientific Ltd</td>
			<td>BPE359-500</td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Sigma-Aldrich</td>
			<td>S8750-500G</td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Fisher Scientific Ltd</td>
			<td>A/0420/PB17</td>
		</tr>
		<tr>
			<td>Novozyme 188</td>
			<td>Novozyme</td>
			<td>DCN00214</td>
		</tr>
		<tr>
			<td>Celluclast 1.5L</td>
			<td>Novozyme</td>
			<td>CCN03122</td>
		</tr>
		<tr>
			<td>96 well PCR full skirt plates</td>
			<td>Sarstedt Ltd</td>
			<td>72.1980.202</td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Fisher Scientific Ltd</td>
			<td>G/0450/53</td>
		</tr>
		<tr>
			<td>3-Methyl-2-benzothiazolinone hydrazone hydrochloride hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>129739-25G</td>
		</tr>
		<tr>
			<td>DL-dithiothreitol</td>
			<td>Sigma-Aldrich</td>
			<td>D9163-1G</td>
		</tr>
		<tr>
			<td>Corning microplate 96 well flat bottom </td>
			<td>Fisher Scientific Ltd</td>
			<td>TKT-521-050H</td>
		</tr>
		<tr>
			<td>Ammonium iron (III) sulfate dodecahydrate </td>
			<td>Sigma-Aldrich</td>
			<td>F1668-250G</td>
		</tr>
		<tr>
			<td>Sulfamic acid</td>
			<td>Sigma-Aldrich</td>
			<td>242772-500G</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Fisher Scientific Ltd</td>
			<td>12462-0026</td>
		</tr>
	</tbody>
</table>

high-throughput saccharification assay for lignocellulosic materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

Watch this Scientific Journal Video about High-throughput Saccharification Assay for Lignocellulosic Materials at JoVE.com

High-throughput Saccharification Assay for Lignocellulosic Materials

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

high-throughput-saccharification-assay-for-lignocellulosic-materials

Research

JoVE Journal

Biology

13.2K 次观看.  University of York. 描述一个简单，快速的方法确定了大量的植物生物量的样品糖化潜力。这种分析的自动化平台，涉及植物生物量在96孔板和随后的表现预处理，水解和释放糖的定量分析的准备。

视频: High-throughput Saccharification Assay for Lignocellulosic Materials

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 ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

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 ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Detection of miRNA Targets in High-throughput Using the 3&#039;LIFE Assay

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...