Name: nanodrop microvolume quantitation of nucleic acids
Uploaded: 2010-11-22T17:00:00.000+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.

<table><tbody><tr><td>NanoDrop 2000c</td><td>Thermo Fisher Scientific, Inc.</td><td>ND-2000C</td><td></td></tr><tr><td>NanoDrop 3300</td><td>Thermo Fisher Scientific, Inc.</td><td>ND-3300</td><td></td></tr></tbody></table>

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

Nanyang Technological University

Research

JoVE Journal

Biology

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Video: NanoDrop Microvolume Quantitation of Nucleic Acids

Methylated DNA Immunoprecipitation

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on gene expression, and is involved with normal development as well as disease 1-4. Thus, the ability to discriminate between methylated DNA and non-methylated DNA is essential for generating methylation profiles for such studies. Methylated DNA immunoprecipitation (MeDIP) is an efficient technique for the extraction of methylated DNA from a sample of interest 5-7. A sample of as little as 200 ng of DNA is sufficient for the antibody, or immunoprecipitation (IP), reaction. DNA is sonicated into fragments ranging in size from 300-1000 bp, and is divided into immunoprecipitated (IP) and input (IN) portions. IP DNA is subsequently heat denatured and then incubated with anti-5'mC, allowing the monoclonal antibody to bind methylated DNA. After this, magnetic beads containing a secondary antibody with affinity for the primary antibody are added, and incubated. These bead-linked antibodies will bind the monoclonal antibody used in the first step. DNA bound to the antibody complex (methylated DNA) is separated from the rest of the DNA by using a magnet to pull the complexes out of solution. Several washes using IP buffer are then performed to remove the unbound, non-methylated DNA. The methylated DNA/antibody complexes are then digested with Proteinase K to digest the antibodies leaving only the methylated DNA intact. The enriched DNA is purified by phenol:chloroform extraction to remove the protein matter and then precipitated and resuspended in water for later use. PCR techniques can be used to validate the efficiency of the MeDIP procedure by analyzing the amplification products of IP and IN DNA for regions known to lack and known to contain methylated sequences. The purified methylated DNA can then be used for locus-specific (PCR) or genome-wide (microarray and sequencing) methylation studies, and is particularly useful when applied in conjunction with other research tools such as gene expression profiling and array comparative genome hybridization (CGH) 8. Further investigation into DNA methylation will lead to the discovery of new epigenetic targets, which in turn, may be useful in developing new therapeutic or prognostic research tools for diseases such as cancer that are characterized by aberrantly methylated DNA 2, 4, 9-11.

This video demonstrates the protocol for methylated DNA immunoprecipitation (MeDIP). MeDIP is a two day procedure that selectively extracts methylated DNA fragments from a genomic DNA sample using antibodies with specificity for 5 -methylcytosine (anti-5 mC).

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on ...

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 ...

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been linked to a disease state and has the potential to be used as disease biomarker. A sensitive and accurate method for circulating microRNA quantification using a dye-based chemistry and droplet digital PCR technology has been recently developed. Specifically, using Locked Nucleic Acid (LNA)-based miRNA-specific primers with a green fluorescent DNA-binding dye in a compatible droplet digital PCR system it is possible to obtain the absolute quantification of specific miRNAs. Here, we describe how performing this technique to assess miRNA amount in biological fluids, such as plasma and serum, is both feasible and effective.

A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been ...

Bioengineering

Precision DNAzyme Nanomachines for Discriminative RNA and DNA Analysis

DNAzyme 10-23 - Based Nanomachines for Nucleic Acid Recognition

DNAzyme-based nanomachines (DNM) for the detection of DNA and RNA sequences (analytes) are multifunctional structures made of oligonucleotides. Their functions include tight analyte binding, highly selective analyte recognition, fluorescent signal amplification by multiple catalytic cleavages of a fluorogenic reporter substrate, and fluorogenic substrate attraction for an increase in sensor response. Functional units are attached to a common DNA scaffold for their cooperative action. The RNA-cleaving 10-23 DNMs feature&#160;improved sensitivity in comparison with non-catalytic hybridization probes. The stability of the DNM and the increased chances of substrate recognition are provided&#160;by a double-stranded DNA fragment,&#160;a tile. DNM can differentiate two analytes with a single nucleotide difference in a folded RNA and a double-stranded DNA and detect analytes at concentrations ~1000 times lower than other protein-free hybridization probes. This article presents the concept behind the diagnostic potential of DNA-nanomachine activity and overviews DNM design, assembly, and application&#160;in nucleic acid detection assays.

Author Spotlight: Advancements in DNA Nanosensors &#8211; Addressing Sensitivity and Selectivity Challenges in Molecular Detection

The DNAzyme-based nanomachines can be used for highly selective and sensitive detection of nucleic acids. This article describes a detailed protocol for the design of DNAzyme-based nanomachines with a 10-23 core using free software and their application in the detection of an Epstein-Barr virus fragment as an example.

DNAzyme-based nanomachines (DNM) for the detection of DNA and RNA sequences (analytes) are multifunctional structures made of oligonucleotides. Their ...

Biochemistry

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 ...

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 ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

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 ...

Measuring Single-Cell Mitochondrial DNA Copy Number and Heteroplasmy Using Digital Droplet Polymerase Chain Reaction

The mammalian mitochondrial (mt)DNA is a small, circular, double-stranded, intra-mitochondrial DNA molecule, encoding 13 subunits of the electron transport chain. Unlike the diploid nuclear genome, most cells contain many more copies of mtDNA, ranging from less than 100 to over 200,000 copies depending on cell type. MtDNA copy number is increasingly used as a biomarker for a number of age-related degenerative conditions and diseases, and thus, accurate measurement of the mtDNA copy number is becoming a key tool in both research and diagnostic settings. Mutations in the mtDNA, often occurring as single nucleotide polymorphisms (SNPs) or deletions, can either exist in all copies of the mtDNA within the cell (termed homoplasmy) or as a mixture of mutated and WT mtDNA copies (termed heteroplasmy). Heteroplasmic mtDNA mutations are a major cause of clinical mitochondrial pathology, either in rare diseases or in a growing number of common late-onset diseases such as Parkinson's disease. Determining the level of heteroplasmy present in cells is a critical step in the diagnosis of rare mitochondrial diseases and in research aimed at understanding common late-onset disorders where mitochondria may play a role. MtDNA copy number and heteroplasmy have traditionally been measured by quantitative (q)PCR-based assays or deep sequencing. However, the recent introduction of ddPCR technology has provided an alternative method for measuring both parameters. It offers several advantages over existing methods, including the ability to measure absolute mtDNA copy number and sufficient sensitivity to make accurate measurements from single cells even at low copy numbers. Presented here is a detailed protocol describing the measurement of mtDNA copy number in single cells using ddPCR, referred to as droplet generation PCR henceforth, with the option for simultaneous measurement of heteroplasmy in cells with mtDNA deletions. The possibility of expanding this method to measure heteroplasmy in cells with mtDNA SNPs is also discussed.

Here we present a protocol for measuring absolute mitochondrial (mt)DNA copy number and mtDNA deletion heteroplasmy levels in single cells.

The mammalian mitochondrial (mt)DNA is a small, circular, double-stranded, intra-mitochondrial DNA molecule, encoding 13 subunits of the electron ...

Medicine

Quantification of Circular RNAs Using Digital Droplet PCR

Digital droplet polymerase chain reaction (dd-PCR) is one of the most sensitive quantification methods; it fractionates the reaction into nearly 20,000 water-in-oil droplets, and the PCR occurs in the individual droplets. The dd-PCR has several advantages over conventional real-time qPCR, including increased accuracy in detecting low-abundance targets, omitting reference genes for quantification, eliminating technical replicates for samples, and showing high resilience to inhibitors in the samples. Recently, dd-PCR has become one of the most popular methods for accurately quantifying target DNA or RNA for gene expression analysis and diagnostics. Circular RNAs (circRNAs) are a large family of recently discovered covalently closed RNA molecules lacking 5' and 3' ends. They have been shown to regulate gene expression by acting as sponges for RNA-binding proteins and microRNAs. Furthermore, circRNAs are secreted into body fluids, and their resistance to exonucleases makes them serve as biomarkers for disease diagnosis. This article aims to show how to perform divergent primer design, RNA extraction, cDNA synthesis, and dd-PCR analysis to accurately quantify specific circular RNA (circRNA) levels in cells. In conclusion, we demonstrate the precise quantification of circRNAs using dd-PCR.

This protocol describes the detailed method of digital droplet PCR (dd-PCR) for precise quantification of circular RNA (circRNA) levels in cells using divergent primers.

Digital droplet polymerase chain reaction (dd-PCR) is one of the most sensitive quantification methods; it fractionates the reaction into nearly 20,000 ...