Name: quantitative real-time pcr using the thermo scientific solaris qpcr assay
Uploaded: 2010-06-17T17:04:00
Duration: 9 min 21 s
Description: Watch this Scientific Journal Video about Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay at JoVE.com

1. The Thermo Scientific Solaris qPCR Assay Design Algorithm
<ol>
 <li>The algorithm used in the design of Solaris qPCR probe/primer pairs adjusts the Tm of each component thereby enabling universal cycling conditions. Incorporation of the MGB, or minor groove binding, moiety in the probe increases the Tm and allows for shorter probes to be designed for greater assay flexibility and performance. As their name implies, MGBs are a class of antibiotics that can fit into the minor groove of the DNA helix. MGBs are attached to the 3 or 5 end of a DNA probe. In solution, the FAM reporter fluorescence of the probe is quenched by Eclipse Dark Quencher. When the probe binds to a target sequence, the resulting change in the probe s 3-D conformation allows a strong fluorescent signal. Binding of the DNA probe to target sequence is stabilized by the MGB, which allows the use of highly specific, shorter probes while maintaining the appropriate melting temperature for PCR. Thus, signal detection occurs during the annealing phase of target amplification. During the extension step, the MGB probe disassociates and the fluorescence is again quenched. Because the probe disassociates, it does not affect PCR efficiency.</li>
 <li>Another feature of the Solaris algorithm is the use of Superbases. Superbases are modified bases that have enhanced binding abilities to improve stability of A-T bonds and eliminate G-G self-association in guanine rich sequences. Moreover, they do not quench adjacent fluorophores attached at the 5 terminus. These bases are selectively placed, according to the algorithm, to expand the availability of target sequences. This makes it possible to use sequences, such as G-C rich regions, that would otherwise be avoided in designing primers and probes. </li>
 <li>Whenever possible, the Solaris algorithm incorporates exon junction spanning regions in the target site. This increases specificity by avoiding the amplification of contaminant genomic DNA. </li>
 <li>Importantly, the algorithm also identifies a consensus sequence for all known splice variants, such that all are covered by a single probe/primer set. So, only one assay is needed for a particular gene of interest. </li>
 <li>Finally, the Solaris qPCR Assay design algorithm utilizes very stringent parameters for critical design considerations including GC content, length, Tm, and stretches of homogenous nucleotides. The resulting sequences are BLAST analyzed for specificity using 3 different databases: genomic, transcript, and pseudogene. </li>
</ol>
2. Obtaining Solaris Reagents
<ol>
 <li>To obtain the appropriate Solaris reagents, visit <a href="http://dharmacon.com">www.thermo.com/solaris</a></li>
 <li>Once at the website click on "GENEius Product Search". To go directly to the GENEius product search, go to <a href="http://www.dharmacon.com/catalogsearch/consolidatedsearch.aspx">www.thermo.com/SolarisSearch</a> </li>
 <li>Click in the search inquiry box and choose the appropriate target organism. </li>
 <li>The GENEius product search can process many different gene identifiers. Here, enter one of the suggested search terms to find the gene of interest, including Ref Seq Accession, the Gene ID, the Gene Symbol, a Gene Description, the Sanger ID or Accession number, or a catalog number. Then, click on "Search". </li>
 <li>Identify the gene of interest and click on "Select" on the left hand side. This will open a window that shows the different Thermo Scientific products available for the gene of interest. Under "Products" select the "qPCR Detection" Tab. </li>
 <li>Under the "Solaris Gene Expression Assay" heading, you will see one optimally designed Solaris probe/primer assay. This one assay will cover all known splice variants of your gene target. Choose the desired quantity. They are available in 100, 200, 400, and 
 1000 X 25 &mu;l reactions (if the assay is identified as made-to-order, the 100 pack size will not be available). Click on "Add to Cart".</li>
 <li>Next, select the appropriate master mix for the thermal cycler that will be used. To determine which master mix will work best with the machine available, click on "Find out which Master Mix works best with your PCR Cycler!" </li>
 <li>Find the qPCR cycler that will be used on the menu to determine which mix to use. </li>
 <li>Return to the GENEius product search window, and select the appropriate master mix. The master mix comes in 100, 200, 400, and 1000 reaction sizes. Click "Add to Cart".</li>
 <li>Suggested reference genes and verso cDNA synthesis kits can also be ordered for a complete qRT-PCR analysis.</li>
 <li>When the Solaris qPCR reagents arrive, store them at -20&deg;C until ready for use. The following reagents should be received: A 20X solution containing two primers provided at 800 nM each and one gene specific probe provided at 200 nm, sequence information for both the probe and the primers, and Solaris qPCR Gene Expression Master Mix at a 2X concentration. Solaris qPCR Gene Expression Assays are stable for a minimum of 12 months. Avoid repeated freeze thawing. </li>
</ol>
3. qPCR setup
<ol>
 <li>Begin the qPCR protocol with cDNA. Total RNA should be extracted using a spin column method. The Thermo Scientific Verso cDNA synthesis kit is recommended for reverse transcription to generate cDNA. </li>
 <li>Prepare for qPCR by thawing the primers, probes, and master mix on ice. Also have on hand PCR grade water and opaque white qPCR plates, which can improve the sensitivity of real-time PCR. </li>
 <li>The master mix contains all components for real time PCR including: 1) Thermo-Start DNA polymerase, a hot-start version of Thermoprime DNA polymerase. This type of polymerase is used to prevent non-specific amplification during the reaction-set up and requires activation step at 95&deg;C 15 min. 2) dNTPs in the Solaris Master mix, dTTP replaces dUTP to maximize amplification efficiency 3)Proprietary reaction buffer, optimized to work with Solaris primer/probe assays. This reaction buffer contains blue dye to increase contrast between reagent and plastic. 4)ROX, if your qPCR instrument requires ROX. </li>
 <li>Once the solutions have thawed, mix by flicking the tube (do not vortex). Spin them down to prevent loss of reagent.</li>
 <li>Next, prepare a 15 mL sterile Falcon tube to set up the reaction mix for the gene of interest, plus one for the reference gene, scaling up depending on the number of samples. Prepare enough such that three replicates can be prepared for each sample, including a no template control (NTC) and standard curve samples, if the standard curve method will be used for analysis.</li>
 <li>For each sample run on a 96 well plate, mix the following: 5 &mu;L of the 2X Solaris qPCR Master Mix, .5 &mu;l of the Solaris Primer/Probe set (20X), and PCR grade water such that the final reaction volume, once the cDNA is added will be 25 &mu;L. For each sample run on a 384 well plate mix the following: 12.5 &mu;L of the 2X Solaris qPCR Master Mix, 1.25 &mu;l of the Solaris Primer/Probe set (20X), and PCR grade water such that the final reaction volume, once the cDNA is added will be 10 &mu;L. </li>
 <li>Using a multi channel pipettor, transfer the master mix to the appropriate wells of the plate. The blue dye included in the master mix will aid in tracking pipetting progress on the white plates. Then, add the 1-5 &mu;L cDNA template (for a 96 well plate) or 1-2 &mu;L of cDNA template (for a 384 well plate). Pipette up and down to mix. Place the plate in a centrifuge and spin down to remove any bubbles, as these will interfere with the fluorescence readings. </li>
 <li>Program the thermal cycler: The enzyme must be activated with a 95&deg;C 15 min (1 cycle). Then 40 cycles of 95&deg;C, denaturation for 15 seconds, and annealing and extension at 60&deg;C for 60 seconds. </li>
 <li>Place the plate in the qPCR instrument. Start the program. </li>
</ol>
Representative qPCR results using Solaris
<img src="/files/ftp_upload/1700/1700fig1.jpg" alt="Figure 1" /> Figure 1. Solaris assays give reliable detection even at very low input concentrations, as judged by the PCR efficiency and r2 values. Ten 10-fold dilutions of cDNA synthesized from synthetic RNA amplicon sequence or DNA amplicon sequence was amplified on an ABI 7900HT instrument using Solaris qPCR Gene Expression Assay for F2RL1 or CDC20, respectively. The log-scale amplification curves and standard curves are shown along with the performance of each assay including efficiency, r2 value, dynamic range out of 10 log10 dilutions, and the lower limit of detection.
<img src="/files/ftp_upload/1700/1700fig2.jpg" alt="Figure 2" /> Figure 2. Solaris Assays give consistent results even between different researchers and laboratories. siRNAs targeting ALDOA and PPIB, as well as a Non-targeting Control (NTC), were transfected into HeLa cells at 100, 10, 1 and 0.1 nM final concentrations. Cells were harvested and total RNA isolated 48 hrs post-treatment. cDNA was synthesized using Thermo Scientific Verso cDNA Synthesis Kit. An aliquot of the cDNA was amplified in two geographically separated laboratories using Solaris qPCR Gene Expression Assays for detection of ALDOA, PPIB, and GAPDH on a Roche LightCycler 480 (384-well) platform. Knockdown was calculated using the &Delta;&Delta;Cq method (normalized to GAPDH reference gene and NTC-treated cells). The same levels of knockdown were demonstrated for both gene targets between both researchers at both sites.

The authors of this article are employees of Thermo Scientific.

Thermo Scientific Solaris qPCR Assays provide a simple, improved method for performing qPCR analysis. Using a high stringency algorithm, incorporating MGB technology and Superbases, these primer and probe sets maximize the fidelity and robustness of qPCR experiments. One primer and probe set recognizes all known splice variants of a given gene. The target site, whenever possible, includes exon spanning regions, to ensure that genomic DNA is not amplified. These features make it possible to run numerous samples simultaneously, greatly reducing the amount of time needed to perform complex experiments. Since the master mix is blue, it is possible to use white opaque plates, which improve the sensitivity of the qPCR, without losing track of pipetting.
Taken all together, the design features of the Solaris qPCR Assay make this novel qPCR assay a simple, yet high-performance assay that is both specific and robust.

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent / material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Solaris aPCR Gene Expression ROX Master Mix (Intro Pack Size)</td>
 <td>Thermo</td>
 <td><a href="http://www.dharmacon.com/catalog/catalogitemtemplate.aspx?id=4064&amp;imageid=4062" target="_blank">AB-4351/INT</a></td>
 <td>100 x 25 &mu;L rxns</td>
 </tr>
 <tr>
 <td>Solaris qPCR Gene Expression ROX Master Mix</td>
 <td>Thermo</td>
 <td><a href="http://www.dharmacon.com/catalog/catalogitemtemplate.aspx?id=4064&amp;imageid=4062" target="_blank">AB-4351/A</a></td>
 <td>200 x 25 &mu;L rxns</td>
 </tr>
 <tr>
 <td>Solaris qPCR Gene Expression ROX Master Mix</td>
 <td>Thermo</td>
 <td><a href="http://www.dharmacon.com/catalog/catalogitemtemplate.aspx?id=4064&amp;imageid=4062" target="_blank">AB-4351/B</a></td>
 <td>400 x 25 &mu;L rxns</td>
 </tr>
 <tr>
 <td>Solaris qPCR Gene Expression ROX Master Mix</td>
 <td>Thermo</td>
 <td><a href="http://www.dharmacon.com/catalog/catalogitemtemplate.aspx?id=4064&amp;imageid=4062" target="_blank">AB-4351/C</a></td>
 <td>1000 x 25 &mu;L rxns</td>
 </tr>
 <tr>
 <td>Solaris qPCR Gene Expression Assays</td>
 <td>Thermo</td>
 <td>AX-XXXXXX-XX-XXXX*</td>
 <td>100 rxns (1 x 125 &mu;L) 
 200 rxns (2 x 125 &mu;L) 
 400 rxns (4 x 125 &mu;L) 
 or 1000 rxns (10 x 125 &mu;L)
 </td>
 </tr>
 <tr>
 <td>Verso cDNA Synthesis Kit</td>
 <td>Thermo</td>
 <td><a href="http://www.dharmacon.com/product/productlandingtemplate.aspx?id=1177" target="_blank">AB-1453/A</a> 
 <a href="http://www.dharmacon.com/product/productlandingtemplate.aspx?id=1177" target="_blank">AB-1453/B</a></td>
 <td>40 x 20 &mu;L rxns 
100 x 20 &mu;L rxns</td>
 </tr>
 <tr>
 <td>ABgene 96-Well Plate (Skirted, Low Profile, White) **</td>
 <td>Thermo</td>
 <td><a href="http://www.dharmacon.com/Product/ProductLandingTemplate.aspx?id=2084" target="_blank">AB-0800/W</a></td>
 <td>25 plates</td>
 </tr>
 <tr>
 <td>ABgene Diamond Ultra 384-Well PCR Plate, White **</td>
 <td>Thermo</td>
 <td><a href="http://www.dharmacon.com/Product/ProductLandingTemplate.aspx?id=2166" target="_blank">AB-2150/W</a></td>
 <td>25 plates</td>
 </tr>
 <tr>
 <td>Absolute qPCR Adhesive Seal</td>
 <td>Thermo</td>
 <td><a href="http://www.dharmacon.com/Product/ProductLandingTemplate.aspx?id=2008" target="_blank">AB-1170</a></td>
 <td>50 sheets</td>
 </tr>
 </tbody>
</table>
*Catalog number is product specific. Please refer to <a href="http://www.thermo.com/solaris" target="_blank">www.thermo.com/solaris</a> to select the appropriate assay
**For cycler compatibility and color choices, see our lates catalog or visit <a href="http://www.thermo.com/pcrplastics" target="_blank">www.thermo.com/pcrplastics</a>

quantitative real-time pcr using the thermo scientific solaris qpcr assay

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and accuracy of the assay. These primer/probe sets are pre-designed to &gt;98% of the human and mouse genomes and feature significant improvements from previously available technologies. These improvements were made possible by virtue of a novel design algorithm, developed by Thermo Scientific bioinformatics experts.
Several convenient features have been incorporated into the Solaris qPCR Assay to streamline the process of performing quantitative real-time PCR. First, the protocol is similar to commonly employed alternatives, so the methods used during qPCR are likely to be familiar. Second, the master mix is blue, which makes setting the qPCR reactions easier to track. Third, the thermal cycling conditions are the same for all assays (genes), making it possible to run many samples at a time and reducing the potential for error. Finally, the probe and primer sequence information are provided, simplifying the publication process.
Here, we demonstrate how to obtain the appropriate Solaris reagents using the GENEius product search feature found on the ordering web site (www.thermo.com/solaris) and how to use the Solaris reagents for performing qPCR using the standard curve method.

Watch this Scientific Journal Video about Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay at JoVE.com

Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay

The Solaris qPCR Gene Expression Assays are novel pre-designed qPCR primer/probe combinations designed to simplify the qPCR process without sacrificing the specificity and robustness of the assay.

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and ...

quantitative-real-time-pcr-using-thermo-scientific-solaris-qpcr

Research

JoVE Journal

Biology

Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer

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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 ...

Analysis of Gene Expression in Emerald Ash Borer (Agrilus planipennis) Using Quantitative Real Time-PCR

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Analysis of Gene Expression in Emerald Ash Borer Agrilus planipennis Using Quantitative Real Time-PCR

Quantitative real-time PCR (qRT-PCR) is an effective tool to diagnose mRNA levels in different insect tissues and developmental stages. In this report we show the use of qRT-PCR to ascertain mRNA levels in different larval tissues and developmental stages of the invasive insect species, emerald ash borer.

Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB ...

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR (qPCR) Arrays

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower detection limit compared to other methods of gene expression profiling while using smaller amounts of input for each assay.  Automated qPCR setup has improved this field by allowing for greater reproducibility.  Its convenient and rapid setup allows for high-throughput experiments, enabling the profiling of many different genes simultaneously in each experiment.  This method along with internal plate controls also reduces experimental variables common to other techniques.  
We recently developed a qPCR assay for profiling of pre-microRNAs  (pre-miRNAs) using a set of 186 primer pairs.  MicroRNAs have emerged as a novel class of small, non-coding RNAs with the ability to regulate many mRNA targets at the post-transcriptional level.  These small RNAs are first transcribed by RNA polymerase II as a primary miRNA (pri-miRNA) transcript, which is then cleaved into the precursor miRNA (pre-miRNA).  Pre-miRNAs are exported to the cytoplasm where Dicer cleaves the hairpin loop to yield mature miRNAs.  Increases in miRNA levels can be observed at both the precursor and mature miRNA levels and profiling of both of these forms can be useful.  There are several commercially available assays for mature miRNAs; however, their high cost may deter researchers from this profiling technique.  Here, we discuss a cost-effective, reliable, SYBR-based qPCR method of profiling pre-miRNAs.  Changes in pre-miRNA levels often reflect mature miRNA changes and can be a useful indicator of mature miRNA expression.  However, simultaneous profiling of both pre-miRNAs and mature miRNAs may be optimal as they can contribute nonredundant information and provide insight into microRNA processing.  Furthermore, the technique described here can be expanded to encompass the profiling of other library sets for specific pathways or pathogens.

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR qPCR Arrays

We will demonstrate the setup and analysis of pre-microRNA 96-well arrays for QPCR using a robot as well as by hand with a Thermo Scientific Matrix multichannel pipette.

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower ...

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.
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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.

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 ...

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, messages are carried by neurotransmitters released from presynaptic terminals and received by receptors on postsynaptic cells. Our previous research in Caenorhabditis elegans has shown that VSM-1 negatively regulates exocytosis. Additionally, analysis of synapses in vsm-1 mutants showed that animals lacking a fully functional VSM-1 have increased synaptic connectivity. Based on these preliminary findings, we hypothesized that C. elegans VSM-1 may play a crucial role in synaptogenesis. To test this hypothesis, double-labeled microarray analysis was performed, and gene expression profiles were determined. First, total RNA was isolated, reversely transcribed to cDNA, and hybridized to the DNA microarrays. Then, in-silico analysis of fluorescent probe hybridization revealed significant induction of many genes coding for members of the major sperm protein family (MSP) in mutants with enhanced synaptogenesis. MSPs are the major component of sperm in C. elegans and appear to signal nematode oocyte maturation and ovulation . In fruit flies, Chai and colleagues 1 demonstrated that MSP-like molecules regulate presynaptic bouton number and size at the neuromuscular junction. Moreover, analysis performed by Tsuda and coworkers 2 suggested that MSPs may act as ligands for Eph receptors and trigger receptor tyrosine kinase signaling cascades. Lastly, real time PCR analysis corroborated that the gene coding for MSP-32 is induced in vsm-1(ok1468) mutants. Taken together, research performed by our laboratory has shown that vsm-1 mutants have a significant increase in synaptic density, which could be mediated by MSP-32 signaling.

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

Genotyping of Plant and Animal Samples without Prior DNA Purification

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors.
PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination1, 2. Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS)3,4 is performed directly from Arabidopsis plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion3. 
Some plant samples tend to be more challenging when using Direct PCR methods as they contain components that interfere with PCR, such as phenolic compounds. In these cases, an additional step to remove the compounds is traditionally required2,5. Here, this problem is overcome by using a quick and easy dilution protocol followed by Direct PCR amplification (Figure 1). Fifteen year-old oak leaves are used as a model for challenging plants as the specimen contains high amounts of phenolic compounds including tannins. 
Gene transfer into mice is broadly used to study the roles of genes in development, physiology and human disease. The use of these animals requires screening for the presence of the transgene, usually with PCR. Traditionally, this involves a time consuming DNA isolation step, during which DNA for PCR analysis is purified from ear, tail or toe tissues6,7. However, with the Thermo Scientific Phire Animal Tissue Direct PCR Kit transgenic mice can be genotyped without prior DNA purification. In this protocol transgenic mouse genotyping is achieved directly from mouse ear tissues, as demonstrated here for a challenging example where only one primer set is used for amplification of two fragments differing greatly in size.

The Direct PCR approach presented here facilitates PCR amplification directly from small amounts of unpurified plant and animal tissue.

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and ...

Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base excision repair (BER) molecular beacons. The substrate (beacon) is comprised of a deoxyoligonucleotide containing a single base lesion with a 6-Carboxyfluorescein (6-FAM) moiety conjugated to the 5'end and a Dabcyl moiety conjugated to the 3' end of the oligonucleotide. The BER molecular beacon is 43 bases in length and the sequence is designed to promote the formation of a stem-loop structure with 13 nucleotides in the loop and 15 base pairs in the stem1,2. When folded in this configuration the 6-FAM moiety is quenched by Dabcyl in a non-fluorescent manner via F&ouml;rster Resonance Energy Transfer (FRET)3,4. The lesion is positioned such that following base lesion removal and strand scission the remaining 5 base oligonucleotide containing the 6-FAM moiety is released from the stem. Release and detachment from the quencher (Dabcyl) results in an increase of fluorescence that is proportionate to the level of DNA repair. By collecting multiple reads of the fluorescence values, real-time assessment of BER activity is possible. The use of standard quantitative real-time PCR instruments allows the simultaneous analysis of numerous samples. The design of these BER molecular beacons, with a single base lesion, is amenable to kinetic analyses, BER quantification and inhibitor validation and is adaptable for quantification of DNA Repair activity in tissue and tumor cell lysates or with purified proteins. The analysis of BER activity in tumor lysates or tissue aspirates using these molecular beacons may be applicable to functional biomarker measurements. Further, the analysis of BER activity with purified proteins using this quantitative assay provides a rapid, high-throughput method for the discovery and validation of BER inhibitors.

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates. The assay yields rates of DNA Repair activity amenable to kinetic analysis and is adaptable for quantification of DNA Repair activity in tissue and tumor lysates or with purified proteins.

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base ...

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and understanding the nature of such interactions is a central step in biological research. Surface Plasmon Resonance (SPR) is a sensitive detection technique for label-free study of bio-molecular interactions in real time. In a typical SPR experiment, one component (usually a protein, termed 'ligand') is immobilized onto a sensor chip surface, while the other (the 'analyte') is free in solution and is injected over the surface. Association and dissociation of the analyte from the ligand are measured and plotted in real time on a graph called a sensogram, from which pre-equilibrium and equilibrium data is derived. Being label-free, consuming low amounts of material, and providing pre-equilibrium kinetic data, often makes SPR the method of choice when studying dynamics of protein interactions. However, one has to keep in mind that due to the method's high sensitivity, the data obtained needs to be carefully analyzed, and supported by other biochemical methods. 
SPR is particularly suitable for studying membrane proteins since it consumes small amounts of purified material, and is compatible with lipids and detergents. This protocol describes an SPR experiment characterizing the kinetic properties of the interaction between a membrane protein (an ABC transporter) and a soluble protein (the transporter's cognate substrate binding protein).

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance SPR

Surface Plasmon Resonance (SPR) is a label-free method for detecting bio-molecular interactions in real time. Herein, a protocol for a membrane protein:receptor interaction experiment is described, while discussing the pros and cons of the technique.

Protein-protein interactions are pivotal to most, if not all, physiological processes, and  understanding the nature of such interactions is a central ...

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 ...

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

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

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

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