This work was supported by grants from the National Institute of Health RO1-GM085130 and National Science Foundation MCB0929394. We thank Dr. Marion Schmidt for her hospitality and for allowing us to film in her laboratory.

1. Preparation of Footprinting Reagents
<ol>
	<li>Prepare a 10x reaction buffer containing 100 mM sodium cacodylate, 1 mM EDTA, and 1 M KCl. Adjust the pH to 7.4. Filter the buffer using a 0.2 &mu;M acetate filter device (Nalgene). Remark: do not pipet RNA directly into 10x buffer.</li>
	<li>Prepare the titration reaction mix for each reaction as indicated in Table 1. The volume of the titration mix (1x buffer and Mg(II) at the desired concentration) should be 90 &mu;l, before adding 10&mu;l of RNA in 1x buffer.</li>
	<li>Prepare a RNAse T1 digestion buffer containing 6.63M Urea, 20mM sodium citrate, 1mM EDTA, 0.25 &mu;g/&mu;l tRNA, 0.025% xylene cyanol, and 0.025% bromphenol blue. This buffer can be stored at 4 &deg;C for up to 6 months.</li>
	<li>The Fenton reaction requires freshly prepared aqueous solutions of 250 mM Fe(NH4)2(SO4)2, 250 mM EDTA, and 500 mM sodium L-ascorbate. The concentrations noted in chapter 1.5 are optimal for equilibrium experiments in buffer such as sodium cacodylate that does not scavenge radicals and diverse divalent ion concentrations.</li>
	<li>Reagents for the peroxidative (1.5.1) and oxidative (1.5.2) footprinting reaction.
	<ol>
		<li>The peroxidative Fenton reaction requires freshly prepared aqueous solutions of 100 mM Fe-EDTA solution, and 50 mM sodium L-ascorbate and 1.5% H2O2. The Fe-EDTA solution is prepared by mixing Fe(NH4)2(SO4)2 with EDTA to have 1.1 times excess of EDTA over iron.</li>
		<li>Just before initiating the oxidative cleavage reaction (3.4.2), create the Fenton reaction mix by combining 2 &mu;l of Fe(NH4)2(SO4)2 (250 mM), 2.2 &mu;l of EDTA (250 mM), 62.5 &mu;l sodium L-ascorbate (500 mM), 22.5 &mu;l 10x reaction buffer and 135.5 &mu;l of H2O to reach the final concentrations in the footprinting reaction mixture (3.4.2) of 0.10 mM, 0.11 mM, and 6.61 mM, respectively.</li>
	</ol>
	</li>
</ol>
2. RNA Preparation for Footprinting Experiment
<ol>
	<li>RNA can be produced by standard in vitro transcription of DNA templates6 or, if shorter than 50 nucleotides, be purchased (e.g. at Integrated DNA Technologies, www.idtdna.com). Follow recommended laboratory cleanliness procedures to maintain sample integrity of RNA during footprinting experiments.7</li>
	<li>Dephosphorylation8 of in vitro transcribed RNA is necessary prior to 32P labeling of the 5' end of RNA. 10 pmol of RNA are end-labeled by kinasing.9 The solution should be cloudy after successful inactivation of phosphatase.</li>
	<li>Purify radioactively labeled RNA using denaturing gel electrophoresis. The RNA is recovered by two subsequent gel extractions using 0.3 M sodium acetate (pH 5.5). RNA is precipitated by mixing 0.5 ml aqueous solution with 1 ml ethanol and subsequent incubation for 1 minute on dry ice. Spin sample at 4 &deg;C, 12500 rpm for 0.5 hr. Discard the supernatant and wash RNA pellet with 70% ethanol. Remove supernatant after additional centrifugation and dry pellet in vacuum.</li>
	<li>Dissolve the 32P-labeled RNA samples in 330 ul of 1x Buffer.Pipet 30 &mu;l of the RNA solution in a reaction tube, precipitate with ethanol and dry RNA in vacuum; keep this tube for generating the reference ladder (step 3.5). Denature the buffered RNA solution by heating at 95 &deg;C for 2 min. Cool the samples to room temperature for 15 min and give a quick spin to bring down the condensates on the lids of the tubes back into solution.</li>
</ol>
3. The Footprinting Experiment
<ol>
	<li>The electrophoresis comb determines the maximum number of data points per analytical gel. We use a 30 well comb. The reference ladder occupies 2 wells and the uncleaved control one. Prepare 27 reaction tubes. The final Mg(II) concentrations should be evenly spaced on a logarithmic scale spanning orders of magnitude around the titration midpoint.</li>
	<li>Separately, incubate the RNA and the Mg(II) solutions at 50&deg;C for 5 min. Mix 10 &mu;l of RNA solution with 90 &mu;l of the corresponding amount of Mg(II) in 1x buffer to reach a final volume of 100 &mu;l. Incubate the solutions for 30 min at 50&deg;C.</li>
	<li>Let the solutions equilibrate at 25&deg;C for 1 hr while folding occurs.</li>
	<li>Peroxidative(3.4.1) or oxidative (3.4.2) hydroxyl radical footprinting reaction.
	<ol>
		<li>Start the peroxidative reaction by placing droplets of 2 &mu;l Fe-EDTA (100 mM), 2 &mu;l sodium L-ascorbate (50 mM) and 2 &mu;l H2O2 (1.5%) separated from each other at the top inside of the reaction tube containing the RNA solution and start the footprinting reaction by vigorous mixing. Stop the reaction after 15 seconds by adding 300 &mu;l cold absolute ethanol. Turn the tubes 3-5 times. Precipitate, wash and dry the pellet as in step 2.4.</li>
		<li>Start the oxidative hydroxyl radical footprintingreaction by adding 5 &mu;l of the freshly prepared Fenton reaction mix (1.6). Incubate for 30 min at 25&deg;C. Add 300 &mu;l of cold absolute ethanol to quench the reaction and mix by turning the tubes 3-5 times. Precipitate, wash and dry the pellet as indicated in step 2.4.</li>
	</ol>
	</li>
	<li>Generate a RNase T1 digest reference sample according to standard procedures10 using the solution prepared in step 1.3.</li>
	<li>Dissolve RNA pellets in 8 &mu;l gel loading dye II (AMBION) and confirm RNA is resuspended by using the Geiger counter.</li>
	<li>Prepare a denaturing, 8% polyacrylamide sequencing gel according to standard protocols.11 Load samples including two references and a control. Separate RNA fragments at 60 W - 75 W for 2.5 hr. Expose dried gel to a storage phosphor screen overnight. Scan phosphor screen with an imaging system for filmless autoradiography, e.g. Storm 865 (GE Healthcare) or Typhoon (GE Healthcare). Transfer gel image file to your computer.</li>
</ol>
4. Data Analysis
<ol>
	<li>The hydroxyl radical reactivity of each nucleotide is determined by quantifying the intensity of each band on the analytical gel. Download, install, and open SAFA, an easy to use open source software for single band fitting and quantification.12, 13 Follow the user manual instructions. Briefly, load the RNA sequence as .txt file followed by the gel picture as .gel file. Adjust band intensities, define the lanes, choose an anchor lane, and perform a gel alignment. Assignment of bands to nucleotides occurs in reference to RNase T1 digestion ladder. Quantify the band integrals and use the normalization/colorplot feature to normalize and assign potentially invariant residues. Save the output as a .txt file.</li>
	<li>SAFA outputs a spreadsheet containing columns representing lanes on the gel and rows representing the integrated band density of the individual bands corresponding to the RNA fragment. First, identify the protection sites which display a noticeable change in solvent accessibility by comparing the protection profile derived from the no Mg(II) sample with the profile of the end point Mg(II) concentration (50 mM in our example). The lower the value, the more protected the nucleotide is against the attack from hydroxyl radicals and vice versa.</li>
	<li>Create folding isotherms from the normalized band integrals of individual or groups of nucleotides versus Mg(II) concentration. The isotherms are individually scaled to fractional saturation by fi = L + (U - L)&bull;<img src="/files/ftp_upload/3244/3244y.jpg" alt="Y" /> where f denotes the integrated density of the band(s) being analyzed, L and U represent the lower and upper limits to the transition and <img src="/files/ftp_upload/3244/3244y.jpg" alt="Y" /> is fractional saturation.14 The data are fit using a non-linear least square analysis program (We use either Origin (OriginLab) or GraphPad (GraphPad Software, Inc.).) to the Hill equation (1), 
	<img alt="Equation 1" src="/files/ftp_upload/3244/3244eq1.jpg" /> 
	where Kd is the equilibrium dissociation constant, [M] corresponds to the concentration of the variable that mediates the folding reaction, Mg(II) in this example, and nH is the Hill coefficient. This procedure scales the transition to fractional saturation, determines its midpoint and provides a phenomenological test of whether a transition is sigmoidal. Isotherms derived from sequencing gels (Figure 3A) that were analyzed by SAFA (Figure 3B) and fit as described above are shown in Figure 3C. The fractional saturation isotherms shown in Figure 3 were generated by scaling in a spreadsheet the values generated by SAFA against the best fit upper and lower limits (U and L) using the equation fraction = value /(U - L) - L /(U - L).</li>
</ol>
5. Representative Results:
Figure 3 shows representative results from P4-P6 RNA &bull;OH footprinting experiments. The gel image (left) indicates that a) background cleavage is minimal (right lane), b) single nucleotide assignment is possible due to well defined bands in the T1 lane (RNase T1 cleaves at each G), and c) the hydroxyl radical induced fragmentation of RNA is well above background. The transition from low to high Mg(II) is affiliated with decreasing integrated band density of individual and groups of bands indicating formation of RNA tertiary structure. Single or groups of contiguous nucleotide whose cleavage changes concomitantly are referred to as a 'protection'. The &bull;OH protections characteristic of the Mg(II) mediated folding of the P4-P6 RNA closely correspond to the solvent inaccessible regions of the molecule observed in its crystal structure.15
Folded RNA molecules can have very different extents (i.e., how close to background the band densities decline) of tertiary contact protection that have not been correlated with clear structural or dynamic phenomenon. Thus, some RNA molecules will show well defined structural transitions, as shown in Fig 3, and some will not. Band intensities are quantified and normalized by SAFA12 analysis (Figure 3B). The output is a 'thermal' plot visualizing the relative degree of protection against &bull;OH. The color transition describes the accessibility change upon Mg(II) addition. White to red or blue shows more accessible or more protected nucleotides, respectively. Each degree of shading is affiliated with a numerical value which can be plotted as a protection curve (Figure 3C) and analyzed by a binding model such as the Hill equation (1). The equilibrium dissociation constant affiliated with Protection 153-155 is roughly twice that of the corresponding value of Protection 163-164.
<img alt="Figure 1" src="/files/ftp_upload/3244/3244fig1.jpg" /> 
Figure 1. Hydroxyl radical production and P4-P6 RNA folding. A) Fe(II) catalyzes the generation of hydroxyl radicals from hydrogen peroxide. Ascorbate reduces Fe(III) back to ferrous iron. B) In the absence of Mg(II), no P4-P6 tertiary structure is formed, allowing hydroxyl radicals to penetrate and cleave all accessible backbone positions. Addition of the Mg(II) initiates folding of P4-P6, allowing only the solvent accessible backbone to be cleaved by the hydroxyl radicals.
<img alt="Figure 2" src="/files/ftp_upload/3244/3244fig2.jpg" /> 
Figure 2. Outline for a hydroxyl radical footprinting experiment. A) Dephophorylation and 32P 5'-end labeling of RNA. B) Purification of 32P-labeled RNA on a denaturing polyacrylamide gel. C) Excision of RNA band, subsequent RNA extraction, and ethanol precipitation. D) Prefolding and folding of RNA. E) Addition of freshly-prepared Fenton reaction mixture to generate the hydroxyl radicals. F) RNA fragment separation by denaturing polyacrylamide gel electrophoresis. G) Quantitation of RNA fragments by SAFA software.
<img alt="Figure 3" src="/files/ftp_upload/3244/3244fig3.jpg" /> 
Figure 3. Analysis of RNA fragments after peroxidative Fenton footprinting reaction. (A) The RNA was exposed to hydroxyl radicals and the cleavage products were resolved using denaturing polyacrylamide gel electrophoresis (PAGE). The picture shows peroxidative cleavage products with increasing concentration of Mg(II). Reference and control lanes are labeled. This image file is the input for the SAFA program which quantitates the volume of each band. (B) The 'thermal' plot generated by SAFA. (C) A spreadsheet of values relating integrated band density to nucleotide number output from SAFA is analyzed by a binding model such as the Hill equation (1). Regions of protection were chosen according to the increase in band integral density as a function of Mg(II) concentration. The Kd is the Mg(II)concentration at which half of the RNA is folded at the site being monitored. The Hill coefficient, nH, is a measure of the slope of the curve which gives information on the binding cooperativity and provides a lower estimate for the number of magnesium ions involved in folding of this particular site.
<img alt="Table 1" src="/files/ftp_upload/3244/3244table1.jpg" /> 
Table 1. Representative volumes for generating RNA samples containing different Mg(II) concentrations.

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No conflicts of interest declared.

Hydroxyl radical footprinting is a valuable tool to assess the solvent accessible surface area of nucleic acids. Qualitative and quantitative formation of tertiary structure14 can be followed as a function of parameters such as ion type and concentration, pH, temperature, binding proteins or folding co-factors. The compelling combination of a straight forward and inexpensive protocol and the resulting solvent accessibility and folding information on a single-nucleotide level makes this method very attractive. ...

<table border="1">
	<tbody>
		<tr>
			<th>Name</th>
			<th>Company</th>
			<th>Cat#</th>
		</tr>
		<tr>
			<td>Sodium Cacodylate
			(Caution! Toxic)	 	
			</td>
			<td>Sigma</td>
			<td>C4945-25g</td>
		</tr>
		<tr>
			<td>EDTA (0.5 M)</td>
			<td>Ambion</td>
			<td>AM9260G</td>
		</tr>
		<tr>
			<td>DEPC treated water</td>
			<td>Ambion</td>
			<td>AM9915G</td>
		</tr>
		<tr>
			<td>Sodium Acetate (3 M)</td>
			<td>Ambion</td>
			<td>AM9740</td>
		</tr>
		<tr>
			<td>MgCl2 (1 M)</td>
			<td>Ambion</td>
			<td>AM9530G</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Ambion</td>
			<td>AM9902</td>
		</tr>
		<tr>
			<td>Sodium Citrate</td>
			<td>Sigma</td>
			<td>W302600</td>
		</tr>
		<tr>
			<td>tRNA</td>
			<td>Sigma</td>
			<td>R-7876</td>
		</tr>
		<tr>
			<td>Sodium-L-ascorbate</td>
			<td>Sigma</td>
			<td>A7631-25g</td>
		</tr>
		<tr>
			<td>Fe(NH4)2(SO4)2 . 6 H2O</td>
			<td>Sigma</td>
			<td>F1543-500g</td>
		</tr>
		<tr>
			<td>RNase T1</td>
			<td>Fermentas</td>
			<td>EN0541</td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide (30%)</td>
			<td>Sigma</td>
			<td>349887</td>
		</tr>
	</tbody>
</table>

monitoring equilibrium changes in rna structure by 'peroxidative' and 'oxidative' hydroxyl radical footprinting

RNA molecules play an essential role in biology. In addition to transmitting genetic information, RNA can fold into unique tertiary structures fulfilling a specific biologic role as regulator, binder or catalyst. Information about tertiary contact formation is essential to understand the function of RNA molecules. Hydroxyl radicals (&bull;OH) are unique probes of the structure of nucleic acids due to their high reactivity and small size.1 When used as a footprinting probe, hydroxyl radicals map the solvent accessible surface of the phosphodiester backbone of DNA1 and RNA2 with as fine as single nucleotide resolution. Hydroxyl radical footprinting can be used to identify the nucleotides within an intermolecular contact surface, e.g. in DNA-protein1 and RNA-protein complexes. Equilibrium3 and kinetic4 transitions can be determined by conducting hydroxyl radical footprinting as a function of a solution variable or time, respectively. A key feature of footprinting is that limited exposure to the probe (e.g., 'single-hit kinetics') results in the uniform sampling of each nucleotide of the polymer.5
In this video article, we use the P4-P6 domain of the Tetrahymena ribozyme to illustrate RNA sample preparation and the determination of a Mg(II)-mediated folding isotherms. We describe the use of the well known hydroxyl radical footprinting protocol that requires H2O2 (we call this the 'peroxidative' protocol) and a valuable, but not widely known, alternative that uses naturally dissolved O2 (we call this the 'oxidative' protocol). An overview of the data reduction, transformation and analysis procedures is presented.

Watch this Scientific Journal Video about Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting at JoVE.com

Monitoring Equilibrium Changes in RNA Structure by &#039;Peroxidative&#039; and &#039;Oxidative&#039; Hydroxyl Radical Footprinting

This protocol describes how to quantify the Mg(II)-dependent formation of RNA tertiary structure by two methods of hydroxyl radical footprinting.

Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting

RNA molecules play an essential role in biology. In addition to transmitting genetic information, RNA can fold into unique tertiary structures fulfilling ...

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14.2K Views.  Hunter College. This protocol describes how to quantify the Mg(II)-dependent formation of RNA tertiary structure by two methods of hydroxyl radical footprinting.

Video: Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein interactions and more general lipid bilayer-protein interactions. These interactions are particularly important in pharmacological research, as many current pharmaceuticals on the market can alter the lipid bilayer material properties, which can lead to altered membrane protein function.   The formation of gramicidin channels are dependent on conformational changes in gramicidin subunits which are in turn dependent on the properties of the lipid.  Hence the gramicidin channel  current is a reporter of altered properties of the bilayer due to certain compounds.  

Membrane protein function is regulated by the cell membrane lipid composition. This video-article details how to form a patch using bilayer patch electrodes, as well as how to use gramicidin channels as reporters of altered membrane properties.

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein ...

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

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 ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and overall cell responsiveness to ligands and is, itself, influenced by intra- and extracellular conditions, including ligand-induced signaling. Optimized for use with monolayer-plated cultured cells, but extendable to free-floating tissue slices, this protocol uses immunolabelling and colocalizational analysis to track changes in intracellular receptor trafficking following both chronic/prolonged and acute interventions, including exogenous drug treatment. After drug treatment, cells are double-immunolabelled for the receptor and for markers for the intracellular compartments of interest. Sequential confocal microscopy is then used to capture two-channel photomicrographs of individual cells, which are subjected to computerized colocalizational analysis to yield quantitative colocalization scores. These scores are normalized to permit pooling of independent replicates prior to statistical analysis. Representative photomicrographs may also be processed to generate illustrative figures. Here, we describe a powerful and flexible technique for quantitatively assessing induced receptor trafficking.

Receptor trafficking modulates signaling and cell responsiveness to ligands and is, itself, responsive to cell conditions, including ligand-induced signaling. Here, we describe a powerful and flexible technique for quantitatively assessing drug-induced receptor trafficking using immunolabeling and colocalizational analysis.

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...