Name: nanodrop microvolume quantitation of nucleic acids
Uploaded: 2010-11-22T17:00:00
Duration: 9 min 28 s
Description: Watch this Scientific Journal Video about NanoDrop Microvolume Quantitation of Nucleic Acids at JoVE.com

Richard W. Beringer and David L. Ash, Applications Scientists, Thermo Scientific NanoDrop products, for technical assistance and filming.

1. 	Microvolume Nucleic Acid Quantification Using a NanoDrop 2000c Spectrophotometer
<ol>
<li>To begin, clean the upper and lower optical surfaces of the microvolume spectrophotometer sample retention system by pipetting 2 to 3 &mu;L of clean deionized water onto the lower optical surface.</li>
<li>Close the lever arm, ensuring that the upper pedestal comes in contact with the deionized water. Lift the lever arm and wipe off both optical surfaces with a clean, dry, lint-free lab wipe.</li>
<li>Open the NanoDrop software and select the Nucleic Acid application. Use a small-volume, calibrated pipettor to perform a blank measurement by dispensing 1 &mu;L of buffer onto the lower optical surface. Lower the lever arm and select "Blank" in the Nucleic Acid application.</li>
<li>Once the blank measurement is complete, clean both optical surfaces with a clean, dry, lint-free lab wipe.</li>
<li>Choose the appropriate constant for the sample that is to be measured. 
 <table border="1">
 <tbody>
 <tr>
 <td>Sample Type</td>
 <td>Select Option</td>
 <td>Constant Used to Calculate Concentration </td>
 </tr>
 <tr>
 <td>dsDNA</td>
 <td>DNA-50</td>
 <td>50</td>
 </tr>
 <tr>
 <td>ssDNA</td>
 <td>DNA-33</td>
 <td>33</td>
 </tr>
 <tr>
 <td>RNA</td>
 <td>RNA-40</td>
 <td>40</td>
 </tr>
 <tr>
 <td>Oligo</td>
 <td>Custom</td>
 <td>15-150</td>
 </tr>
 </tbody>
 </table>
 Table 1. Typical nucleic acid concentration ranges for direct A280 absorbance measurements using a NanoDrop microvolume spectrophotometer, a traditional cuvette-based spectrophotometer used with a TrayCell microcell cuvette, and a microvolume NanoDrop fluorospectrometer in conjunction with the PicoGreen assay.
</li>
<li>Dispense 1 &mu;L of nucleic acid sample onto the lower optical pedestal and close the lever arm. Because the measurement is volume independent, the sample only needs to bridge the gap between the two optical surfaces for a measurement to be made.</li>
<li>Select "Measure" in the application software. The software will automatically calculate the nucleic acid concentration and purity ratios. Following sample measurement, review the spectral output.</li>
<li>The software will automatically calculate the nucleic acid concentration and purity ratios. Following sample measurement, review the spectral image to assess sample quality.</li>
<li>A typical nucleic acid sample will have a very characteristic profile. 
<img src="/files/ftp_upload/2565/2565fig1.jpg" alt="Figure 1" /> 
Figure 1. A typical nucleic acid sample will have a very characteristic profile.</li>
<li>Common sources of contaminants associated with specific nucleic acid isolation techniques include Phenol/Trizol and column extraction. In the case of Phenol/Trizol extraction, residual reagent contamination may be indicated by abnormal spectra between 220 to 240 nm as well as by shifts in the 260 to 280 nm region. Conversely, residual guanidine from column extraction may contribute to a peak near 230 nm and a shift in the trough from 230 nm to approximately 240 nm. 
<img src="/files/ftp_upload/2565/2565fig2.jpg" alt="Figure 2" /> 
Figure 2. The shifts in the peaks and troughs of samples B and C as compared to sample A illustrate how contaminants can affect the spectra of nucleic acid samples.</li>
<li>To accurately assess sample quality, 260/280 or 260/230 ratios should be analyzed in combination with overall spectral quality. Pure nucleic acids typically yield a 260/280 ratio of ~1.8 and a 260/280 ratio of ~2.0 for DNA and RNA, respectively. This ratio is dependent on the pH and ionic strength of the buffer used to make the blank and sample measurements. Acidic solutions will under-represent the ratio by 0.2-0.3, while a basic solution will over-represent the ratio by 0.2-0.3. Significantly different purity ratios may indicate the presence of protein, phenol or other contaminants that absorb strongly at or near 280 nm. The 260/230 purity ratio is a second measure of DNA purity with values for a "pure" nucleic acid commonly in the range of 1.8-2.2. Purity ratios that are significantly lower than the expected values may indicate the isolation technique used may require further optimization.</li>
<li>NanoDrop spectrophotometers can also be used to quantify proteins using direct absorbance or by protein colorimetric assays. To ensure proper column formation and reproducibility, 2-&mu;l samples are advised for protein samples. Refer to the following JoVE video: Microvolume Protein Concentration Determination Using the NanoDrop Spectrophotometer <a href="https://www.jove.com/index/Details.stp?ID=1610">https://www.jove.com/index/Details.stp?ID=1610</a></li>
</ol>
2. High-Sensitivity Microvolume Nucleic Acid Quantitation Using the NanoDrop 3300 Fluorospectrometer
<ol>
<li>To demonstrate high-sensitivity microvolume nucleic acid quantitation using the NanoDrop 3300, a PicoGreen fluorescence kit is used. To begin, equilibrate the kit standards and all nucleic acid samples to room temperature. Fluorescent samples are light sensitive and should be kept in amber or aluminum wrapped tubes.</li>
<li>Prepare enough 1x TE buffer for all standards and samples to be measured as well as for the PicoGreen working solution that will be needed. Prepare 1xTE buffer by diluting the provided 20x TE buffer with nuclease-free water per the manufacturer's protocol. Reaction volumes can range from 200 &mu;L to as little as 10 ul. In this example, 10 &mu;L reaction volumes will be prepared.</li>
<li>Prepare serially diluted dsDNA standards at 2x the final desired concentration in nuclease-free vials or tubes. Transfer 5 &mu;L of each of the diluted dsDNA standards into an individual labeled nuclease-free amber or foil-covered tube.</li>
<li>Aliquot 5 &mu;L of each dsDNA sample of interest into appropriately labeled nuclease-free amber or foil-covered tubes. </li>
<li>Prepare PicoGreen working solution according to the manufacturer's protocol. Transfer an equal volume of the PicoGreen working solution to each tube containing either a standard or sample of interest (5 &mu;L in this example).</li>
<li>Combine equal volumes of 1x TE buffer and PicoGreen working solution to prepare the negative control, which serves as a reference solution.</li>
<li>Mix the contents of each reaction tube thoroughly by gentle pipetting and incubate the reactions at room temperature for 5 minutes. All standards and samples should be equilibrated to the same temperature before measurement as fluorescence signal is affected by temperature.</li>
<li>To prepare the instrument and perform measurements, first clean the lower and upper surfaces of the sample retention system. Pipet 2 to 3 &mu;L of deionized water onto the lower optical surface, close then open the lever arm, and blot the upper and lower pedestals with a clean, lint-free, laboratory wipe. Ensure the surfaces are free of lint before proceeding as lint can exhibit fluorescence in the presence of an excitation source and can interfere with the measurement.</li>
<li>Open the instrument software, select the "Nucleic Acids" application, and select the "PicoGreen-dsDNA" option.</li>
<li>To blank the instrument, add 2 &mu;L of 1x TE to the lower pedestal, close the arm, and select "Blank" in the instrument software field. Once blank is complete, blot off the blank solution from both surfaces of the sample retention system.</li>
<li>Mix the reference solution by gentle pipetting. Using low retention tips, pipette 2 &mu;L onto the lower pedestal and close the arm.</li>
<li>Select "Reference" in the measurement type, and select the desired units. Click "Measure" to initiate the measurement cycle.</li>
<li>When the measurement cycle is complete, open the arm and thoroughly blot off the sample retention system surfaces. Measure up to five replicates of the reference, using a fresh aliquot for each replicate.</li>
<li>Repeat the process for the additional standards to build a standard curve. Up to seven standards may be used. The software is designed to store up to five replicates for each standard. Replicates of the standards are averaged to generate a standard curve from which the sample concentrations are automatically determined.</li>
<li>Once the standard curve is complete, select the "Samples" tab, enter the respective sample ID information and measure each unknown sample.</li>
</ol>

<ol>
	<li>Wilfinger, W. W., Mackey, K., Chomczynski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+pH+and+Ionic+Strength+on+the+Spectrophotometric+Assessment+of+Nucleic+Acid+Purity.">Effect of pH and Ionic Strength on the Spectrophotometric Assessment of Nucleic Acid Purity.</a> BioTechniques. 22, 474-481 (1997).</li><li>Voolstra, C., Jungnickel, A., Borrmann, L., Kirchner, R., Huber, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrophotometric+Quantification+of+Nucleic+Acids:+LabelGuard+enables+photometric+quantification+of+submicroliter+samples+using+a+standard+photometer.">Spectrophotometric Quantification of Nucleic Acids: LabelGuard enables photometric quantification of submicroliter samples using a standard photometer.</a> Implen Applications Note. , (2006).</li><li>Jr, I. n. g. l. e., D, J., Crouch, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Spectrochemical analysis. , XV-590 (1988).</li><li>Van Lancker, M., Gheyssens, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+four+frequently+used+assays+for+quantitative+determination+of+DNA.">A comparison of four frequently used assays for quantitative determination of DNA.</a> Anal. Lett. 19, 615-623 (1986).</li></ol>

Philippe Desjardins and Debra Conklin are employed by Thermo Scientific NanoDrop Products, Inc that produces the instruments used in this article.

The quantitation protocols presented here are based on the most widely accepted microvolume systems. Such systems provide practical alternatives for traditional nucleic acid quantitation methodology, which rely on greater sample volumes and the use of containment apparatus.
NanoDrop microvolume technology employs a sample retention system that relies on the surface tension properties of the sample being measured to form a liquid column. It is essential that the sample makes contact with the upper and lower optical measurement surfaces for proper column formation. If at any point the measurement results seem inaccurate or not reproducible, it is most likely the result of sample heterogeneity or liquid column breakage.
Detergents and isopropyl alcohol are not recommended cleaning agents as they may uncondition the pedestal measurement surfaces. An unconditioned pedestal occurs when the hydrophobic surface properties of the pedestal have been compromised, resulting in a flattening of the sample droplet. Unconditioned pedestals can lead to breakage of the liquid column during measurement.
<img src="/files/ftp_upload/2565/2565fig3.jpg" alt="Figure 3" /> 
Figure 3. Unconditioned surface is characterized by a flattening of a water droplet.
Uncondtioned pedestal surfaces may be reconditioned using NanoDrop Pedestal Reconditioning Compound (PR-1, Thermo Scientific NanoDrop Products). A thin layer of reconditioning compound is applied to the upper and lower pedestal surfaces, allowed to dry for ~30 seconds, then rubbed off using a clean, dry, lint-free lab wipe. A successfully reconditioned state is characterized by a water droplet beading up when applied to the bottom pedestal surface.
<img src="/files/ftp_upload/2565/2565fig4.jpg" alt="Figure 4" /> 
Figure 4. A reconditioned surface is characterized by a beading up of a water droplet. 
Microvolume quantitation systems greatly reduce sample consumption and dramatically increase the concentration range when compared to more traditional quantification systems. Although microvolume quantitation has often become an enabling technology for circumstances involving limited cell mass such as needle biopsies and laser-capture microdissection, the efficiency and ease-of-use of this methodology has made it a widely accepted alternative to traditional nucleic acid quantitation methods even when sample is plentiful.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>NanoDrop 2000c</td>
<td>&nbsp;</td>
<td>Thermo Fisher Scientific</td>
<td>ND-2000C</td>
<td>&nbsp;</td>
<tr>
<td>NanoDrop 3300</td>
<td>&nbsp;</td>
<td>Thermo Fisher Scientific</td>
<td>ND-3300</td>
<td>&nbsp;</td>		</tbody>
		</table>
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nanodrop microvolume quantitation of nucleic acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.
The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.
The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 &mu;L of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 &mu;L or less are now possible using a dedicated microvolume fluorospectrometer.
Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ &mu;L to 15,000 ng/ &mu;L with minimal consumption of sample.

Watch this Scientific Journal Video about NanoDrop Microvolume Quantitation of Nucleic Acids at JoVE.com

NanoDrop Microvolume Quantitation of Nucleic Acids

The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

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nanodrop-microvolume-quantitation-of-nucleic-acids

Research

JoVE Journal

Biology

Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often used for protein analysis. The Thermo Scientific NanoDrop 2000c Spectrophotometer solves this issue with an innovative sample retention system that holds microvolume samples between two measurement surfaces using the surface tension properties of liquids, enabling the quantification of samples in volumes as low as 0.5-2 &mu;L. The elimination of cuvettes or capillaries allows real time changes in path length, which reduces the measurement time while greatly increasing the dynamic range of protein concentrations that can be measured. The need for dilutions is also eliminated, and preparations for sample quantification are relatively easy as the measurement surfaces can be simply wiped with laboratory wipe. This video article presents modifications to traditional protein concentration determination methods for quantification of microvolume amounts of protein using A280 absorbance readings or the BCA colorimetric assay.

Microvolume samples are quantified by a spectrophotometer system that uses natural surface tension to retain samples without the use of cuvettes or capillaries. The dynamic range of protein concentrations and speed by which they can be measured are greatly increased with this method.

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often ...

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &amp;#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

Nucleic Acid quantitation procedures have advanced significantly in the last three decades. More and more, molecular biologists require consistent small-volume analysis of nucleic acid samples for their experiments. The BioSpec-nano provides a potential solution to the problems of inaccurate, non-reproducible results, inherent in current DNA quantitation methods, via specialized optics and a sensitive PDA detector. The BioSpec-nano also has automated functionality such that mounting, measurement, and cleaning are done by the instrument, thereby eliminating tedious, repetitive, and inconsistent placement of the fiber optic element and manual cleaning.
In this study, data is presented on the quantification of DNA and protein, as well as on measurement reproducibility and accuracy. Automated sample contact and rapid scanning allows measurement in three seconds, resulting in excellent throughput. Data analysis is carried out using the built-in features of the software. The formula used for calculating DNA concentration is:
Sample Concentration = DF &middot; (OD260-OD320)&middot; NACF (1)
Where DF = sample dilution factor and NACF = nucleic acid concentration factor.
The Nucleic Acid concentration factor is set in accordance with the analyte selected1.
Protein concentration results can be expressed as &mu;g/ mL or as moles/L by entering e280 and molecular weight values respectively. When residue values for Tyr, Trp and Cysteine (S-S bond) are entered in the e280Calc tab, the extinction coefficient values are calculated as e280 = 5500 x (Trp residues) + 1490 x (Tyr residues) + 125 x (cysteine S-S bond). The e280 value is used by the software for concentration calculation.
In addition to concentration determination of nucleic acids and protein, the BioSpec-nano can be used as an ultra micro-volume spectrophotometer for many other analytes or as a standard spectrophotometer using 5 mm pathlength cells.

This communication presents data on the accuracy and reproducibility of the BioSpec-nano UV-VIS spectrophotometer for dsDNA and protein quantitation. Even with ultra-small volumes (1 to 2 L), reproducibility is excellent, while the automated wiping function improves throughput and results in minimal carryover for more precise results.

Nucleic Acid quantitation procedures have advanced significantly in the last three decades.  More and more, molecular biologists require consistent ...

Electricity-Free, Sequential Nucleic Acid and Protein Isolation

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable 1. The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment 2. The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis.
Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters 3. CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation4.
By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification while the protein content can immediately be analyzed by hand held or other immunological-based assays. The rapid identification of disease markers in the field could significantly alter the patient's outcome by directing the proper course of treatment at an earlier stage of disease progression. The tool and method described are suitable for use with virtually any infectious agent and offer the user the redundancy of multi-macromolecule type analyses while simultaneously reducing their logistical burden.

A tool and chemistries are described to sequentially isolate nucleic acids followed by proteins from a sample without the need for electricity. The tool consists of a sorbent held within a transfer pipette while the isolation chemistries are based on solid-phase extraction principles. The isolated macromolecules can be analyzed by immuno-based and PCR-based assays.

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern ...

Analyzing and Building Nucleic Acid Structures with 3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures. Protocol 1 lists the set of instructions needed to download and install the software. This is followed, in Protocol 2, by the analysis of a nucleic acid structure, including the assignment of base pairs and the determination of rigid-body parameters that describe the structure and, in Protocol 3, by a description of the reconstruction of an atomic model of a structure from its rigid-body parameters. The most recent version of 3DNA, version 2.1, has new features for the analysis and manipulation of ensembles of structures, such as those deduced from nuclear magnetic resonance (NMR) measurements and molecular dynamic (MD) simulations; these features are presented in Protocols 4 and 5. In addition to the 3DNA stand-alone software package, the w3DNA web server, located at <a href="http://w3dna.rutgers.edu" target="_blank">http://w3dna.rutgers.edu</a>, provides a user-friendly interface to selected features of the software. Protocol 6 demonstrates a novel feature of the site for building models of long DNA molecules decorated with bound proteins at user-specified locations.

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures.

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic ...

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, and participate in various signaling pathways. Caenorhabditis elegans synthesizes all of the enzymes necessary to produce a range of omega-6 and omega-3 fatty acids. This, combined with the simple anatomy and range of available genetic tools, make it an attractive model to study fatty acid function. In order to investigate the genetic pathways that mediate the physiological effects of dietary fatty acids, we have developed a method to supplement the C. elegans diet with unsaturated fatty acids. Supplementation is an effective means to alter the fatty acid composition of worms and can also be used to rescue defects in fatty acid-deficient mutants. Our method uses nematode growth medium agar (NGM) supplemented with fatty acidsodium salts. The fatty acids in the supplemented plates become incorporated into the membranes of the bacterial food source, which is then taken up by the C. elegans that feed on the supplemented bacteria. We also describe a gas chromatography protocol to monitor the changes in fatty acid composition that occur in supplemented worms. This is an efficient way to supplement the diets of both large and small populations of C. elegans, allowing for a range of applications for this method.

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Caenorhabditis elegans is a useful model to explore the functions of polyunsaturated fatty acids in development and physiology. This protocol describes an efficient method of supplementing the C. elegans diet with polyunsaturated fatty acids.

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

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 ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method&#160;applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.
Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...