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><tbody><tr><td>Solaris aPCR Gene Expression ROX Master Mix (Intro Pack Size)</td><td>Thermo Fisher Scientific, Inc.</td><td>&lt;a href=&quot;http://www.dharmacon.com/catalog/catalogitemtemplate.aspx?id=4064&amp;amp;imageid=4062&quot; target=&quot;_blank&quot;&gt;AB-4351/INT&lt;/a&gt;</td><td>100 x 25 &amp;#x03BC;L rxns</td></tr><tr><td>Solaris qPCR Gene Expression ROX Master Mix</td><td>Thermo Fisher Scientific, Inc.</td><td>&lt;a href=&quot;http://www.dharmacon.com/catalog/catalogitemtemplate.aspx?id=4064&amp;amp;imageid=4062&quot; target=&quot;_blank&quot;&gt;AB-4351/A&lt;/a&gt;</td><td>200 x 25 &amp;#x03BC;L rxns</td></tr><tr><td>Solaris qPCR Gene Expression ROX Master Mix</td><td>Thermo Fisher Scientific, Inc.</td><td>&lt;a href=&quot;http://www.dharmacon.com/catalog/catalogitemtemplate.aspx?id=4064&amp;amp;imageid=4062&quot; target=&quot;_blank&quot;&gt;AB-4351/B&lt;/a&gt;</td><td>400 x 25 &amp;#x03BC;L rxns</td></tr><tr><td>Solaris qPCR Gene Expression ROX Master Mix</td><td>Thermo Fisher Scientific, Inc.</td><td>&lt;a href=&quot;http://www.dharmacon.com/catalog/catalogitemtemplate.aspx?id=4064&amp;amp;imageid=4062&quot; target=&quot;_blank&quot;&gt;AB-4351/C&lt;/a&gt;</td><td>1000 x 25 &amp;#x03BC;L rxns</td></tr><tr><td>Solaris qPCR Gene Expression Assays</td><td>Thermo Fisher Scientific, Inc.</td><td>AX-XXXXXX-XX-XXXX*</td><td>100 rxns (1 x 125 &amp;#x03BC;L)&lt;br/&gt; 200 rxns (2 x 125 &amp;#x03BC;L)&lt;br/&gt; 400 rxns (4 x 125 &amp;#x03BC;L)&lt;br/&gt; or 1000 rxns (10 x 125 &amp;#x03BC;L) </td></tr><tr><td>Verso cDNA Synthesis Kit</td><td>Thermo Fisher Scientific, Inc.</td><td>&lt;a href=&quot;http://www.dharmacon.com/product/productlandingtemplate.aspx?id=1177&quot; target=&quot;_blank&quot;&gt;AB-1453/A&lt;/a&gt; &lt;a href=&quot;http://www.dharmacon.com/product/productlandingtemplate.aspx?id=1177&quot; target=&quot;_blank&quot;&gt;AB-1453/B&lt;/a&gt;</td><td>40 x 20 &amp;#x03BC;L rxns&lt;br/&gt;100 x 20 &amp;#x03BC;L rxns</td></tr><tr><td>ABgene 96-Well Plate (Skirted, Low Profile, White) **</td><td>Thermo Fisher Scientific, Inc.</td><td>&lt;a href=&quot;http://www.dharmacon.com/Product/ProductLandingTemplate.aspx?id=2084&quot; target=&quot;_blank&quot;&gt;AB-0800/W&lt;/a&gt;</td><td>25 plates</td></tr><tr><td>ABgene Diamond Ultra 384-Well PCR Plate, White **</td><td>Thermo Fisher Scientific, Inc.</td><td>&lt;a href=&quot;http://www.dharmacon.com/Product/ProductLandingTemplate.aspx?id=2166&quot; target=&quot;_blank&quot;&gt;AB-2150/W&lt;/a&gt;</td><td>25 plates</td></tr><tr><td>Absolute qPCR Adhesive Seal</td><td>Thermo Fisher Scientific, Inc.</td><td>&lt;a href=&quot;http://www.dharmacon.com/Product/ProductLandingTemplate.aspx?id=2008&quot; target=&quot;_blank&quot;&gt;AB-1170&lt;/a&gt;</td><td>50 sheets</td></tr><tr><td colspan="4">*Catalog number is product specific. Please refer to &lt;a href=&quot;http://www.thermo.com/solaris&quot; target=&quot;_blank&quot;&gt;www.thermo.com/solaris&lt;/a&gt; to select the appropriate assay</td></tr><tr><td colspan="4">**For cycler compatibility and color choices, see our lates catalog or visit &lt;a href=&quot;http://www.thermo.com/pcrplastics&quot; target=&quot;_blank&quot;&gt;www.thermo.com/pcrplastics&lt;/a&gt;</td></tr></tbody></table>

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

Universidad San Sebastian

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JoVE Journal

Biology

49.7K Views.  Thermo Scientific Solaris qPCR Products.  日本語 

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

Looking for Driver Pathways of Acquired Resistance to Targeted Therapy: Drug Resistant Subclone Generation and Sensitivity Restoring by Gene Knock-down

The past two decades have seen a shift from cytotoxic drugs to targeted therapy in medical oncology. Although targeted therapeutic agents have shown more impressive clinical efficacy and minimized adverse effects than traditional treatments, drug resistance has become the main limitation to their benefits. Several preclinical in vitro/in vivo models of acquired resistance to targeted agents in clinical practice have been developed mainly by using two strategies: i) genetic manipulation for modeling genotypes of acquired resistance, and ii) in vitro/in vivo selection of resistant models. In the present work, we propose a unifying framework, for investigating the underlying mechanisms responsible for acquired resistance to targeted therapeutic agents, starting from the generation of drug-resistant cellular subclones to the description of silencing procedures used for restoring the sensitivity to the inhibitor. This simple time- and cost-effective approach is widely applicable, and could be easily extended to investigate resistance mechanisms to other targeted therapeutic drugs in different tumor histotypes.

This is a time- and cost-effective in vitro protocol investigating the mechanisms of acquired resistance to targeted therapeutic agents, which is a highly unmet medical need in cancer management.

The past two decades have seen a shift from cytotoxic drugs to targeted therapy in medical oncology. Although targeted therapeutic agents have shown more ...

Cancer Research

On-Site Molecular Detection of Soil-Borne Phytopathogens Using a Portable Real-Time PCR System

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management strategies that reduce the impact of the disease. Presently in many diagnostic laboratories, the polymerase chain reaction (PCR), particularly real-time PCR, is considered the most sensitive and accurate method for plant pathogen detection. However, laboratory-based PCRs typically require expensive laboratory equipment and skilled personnel. In this study, soil-borne pathogens of potato are used to demonstrate the potential for on-site molecular detection. This was achieved using a rapid and simple protocol comprising of magnetic bead-based nucleic acid extraction, portable real-time PCR (fluorogenic probe-based assay). The portable real-time PCR approach compared favorably with a laboratory-based system, detecting as few as 100 copies of DNA from Spongospora subterranea. The portable real-time PCR method developed here can serve as an alternative to laboratory-based approaches and a useful on-site tool for pathogen diagnosis.

Rapid and accurate detection of plant pathogens on-site, especially soil-borne pathogens, is crucial to prevent further inoculum production and proliferation of plant diseases in the field. The method developed here using a portable real-time PCR detection system enables on-site diagnosis under field conditions.

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Environment

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

Medicine

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Immunology and Infection

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 ...

Quantitative PCR of T7 Bacteriophage from Biopanning

This protocol describes the use of quantitative PCR (qPCR) to enumerate T7 phages from phage selection experiments (i.e., &#34;biopanning&#34;). qPCR is a fluorescence-based approach to quantify DNA, and here, it is adapted to quantify phage genomes as a proxy for phage particles. In this protocol, a facile phage DNA preparation method is described using high-temperature heating without additional DNA purification. The method only needs small volumes of heat-treated phages and small volumes of the qPCR reaction. qPCR is high-throughput and fast, able to process and obtain data from a 96-well plate of reactions in 2&#8211;4 h. Compared to other phage enumeration approaches, qPCR is more time-efficient. Here, qPCR is used to enumerate T7 phages identified from biopanning against in vitro cystic fibrosis-like mucus model. The qPCR method can be extended to quantify T7 phages from other experiments, including other types of biopanning (e.g.,&#160;immobilized protein binding, in vivo phage screening) and other sources (e.g., water systems or body fluids). In summary, this protocol can be modified to quantify any DNA-encapsulated viruses.

A reproducible, accurate, and time efficient quantitative PCR (qPCR) method to enumerate T7 bacteriophage is described here. The protocol clearly describes phage genomic DNA preparation, PCR reaction preparation, qPCR cycling conditions, and qPCR data analysis.

This protocol describes the use of quantitative PCR (qPCR) to enumerate T7 phages from phage selection experiments (i.e., &#34;biopanning&#34;). qPCR is a ...

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.

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Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to quantify DNA sample concentration using a standard curve. In this manuscript, a detailed protocol for developing and implementing a real-time quantitative recombinase polymerase amplification assay (qRPA assay) is provided. Using HIV-1 DNA quantification as an example, the assembly of real-time RPA reactions, the design of an internal positive control (IPC) sequence, and co-amplification of the IPC and target of interest are all described. Instructions and data processing scripts for the construction of a standard curve using data from multiple experiments are provided, which may be used to predict the concentration of unknown samples or assess the performance of the assay. Finally, an alternative method for collecting real-time fluorescence data with a microscope and a stage heater as a step towards developing a point-of-care qRPA assay is described. The protocol and scripts provided may be used for the development of a qRPA assay for any DNA target of interest.

Provided is a protocol for developing a real-time recombinase polymerase amplification assay to quantify initial concentration of DNA samples using either a thermal cycler or a microscope and stage heater. Also described is the development of an internal positive control. Scripts are provided for processing raw real-time fluorescence data.

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to ...

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 ...

Quantifying Environmental Microorganisms and Viruses Using qPCR

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona 
Demonstrating Author: Bradley Schmitz
Quantitative polymerase chain reaction (qPCR), also known as real-time PCR, is a widely-used molecular technique for enumerating microorganisms in the environment. Prior to this approach, quantifying microorganisms was limited largely to classical culture-based techniques. However, the culturing of microbes from environmental samples can be particularly challenging, and it is generally held that as few as 1 to 10% of the microorganisms present within environmental samples are detectable using these techniques. The advent of qPCR in environmental microbiology research has therefore advanced the field greatly by allowing for more accurate determination of concentrations of microorganisms such as disease-causing pathogens in environmental samples. However, an important limitation of qPCR as an applied microbiological technique is that living, viable populations cannot be differentiated from inactive or non-living populations.
This video demonstrates the use of qPCR to detect pepper mild mottle virus from an environmental water sample.

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona
Demonstrating Author: Bradley Schmitz
Quantitative polymerase ...