PCR and qPCR
Support and Educational Content

Improved PCR genotyping—obtain greater precision with RNase H2 activation of assay primers

Learn about the core mechanism behind the IDT rhAmp Genotyping System and how it improves on existing 5′-nuclease PCR assay technologies.

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Improve the specificity of SNP genotyping

Webinar summary: This novel, simple genotyping method uses blocked, cleavable primers and RNase H2 to prevent off-target and primer-dimer interactions. Learn how these single-tube reactions, easily run in high-throughput mode, can improve the accuracy and sensitivity of your genotyping experiments.

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LISH method enables highly multiplexed, sensitive gene expression in FFPE samples, without RT

Ligation in situ Hybridization (LISH) enables sensitive, multiplex detection of mRNA expression in archival FFPE samples, without RNA isolation or reverse transcription. The authors show that LISH can be used to detect expression in archived FFPE specimens up to 10 years old and that it can be highly multiplexed (>100 target sequences). Review the mechanism of this technique, and learn about its many applications.

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16S rRNA indexed primers amplify phylogenic markers for microbiome sequencing analysis

The 16S rRNA gene is frequently used in microbiome studies to identify the subset of microbes present in biological samples. Researchers amplify short hypervariable regions from this gene, tag the amplified products with unique barcodes, perform highly multiplexed sequencing runs, and compare the sequences to the known bacterial genome database. However, primer design for such analyses can be challenging given the massive sequence variability in sampled lifeforms. Read about the development and design of these primers, and how you can obtain your own custom, high fidelity versions of these sequences.

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rhPCR SNP genotyping method provides calls with >99.5% accuracy

Use this simple, single-tube genotyping platform to get >99.5% call accuracy. The method is based on RNase H2-dependent PCR technology and uses RNA-DNA hybrid primers, blocked at the 3′ end, that prevent primer-dimer artifacts and non-specific amplification.

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Considerations when adopting published sequences for your own qPCR assay

Lab tip: Published papers that use qPCR applications are a resource of vetted assay sequences. These can be co-opted and converted to more sensitive, double-quenched probes for use in your own experiments. Before ordering, though, go through this checklist to ensure that these sequences will work well in your assays, and consider up-to-date PrimeTime Predesigned qPCR Assays with guaranteed efficiencies of >90%.

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Consider SNPs when designing PCR and qPCR assays

NGS has led to a dramatic increase in identified SNPs. SNPs can pose a problem when they underlie primer or probe sequences used in PCR/qPCR. Learn what effect they can have and how you can minimize their impact on your PCR assays.

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Analyzing microbiomes—their impact on our health and our environment

Research profile: One of the leading experts in microbiome research and founding member of the Human Microbiome Project, Dr Rob Knight looks for connections between the microbiomes of people and places to human and environmental health. Learn how the Knight lab addresses the challenges of DNA isolation and 16S rRNA primer design and validation for the diverse microbiota that comprise these study samples.

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Improve probe-based qPCR experiments

Webinar summary: Learn about the benefits of our PrimeTime® Gene Expression Master Mix for qPCR experiments. Suitable for singleplex and multiplex experiments, online tools for planning qPCR experiments, and a discussion of the excellent thermal stability of the master mix.

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Increasing ddPCR performance in low target HIV assays

ZEN™ Double-Quenched qPCR Probes work well in ddPCR. Learn how Dr Matthew Strain’s lab used ZEN™ Double-Quenched qPCR Probes in ddPCR, to achieve additional sensitivity through lower background in experiments with low copy number samples, where individual droplets matter. The Strain lab has published open-access protocols for their HIV assays that include these updated ZEN probe designs at www.bio-protocol.org.

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Improved pathogen detection by multiplex RT-qPCR

Citation summary: Learn how gBlocks Gene Fragments can help optimize multiplex qPCR for pathogen detection in human clinical samples.

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Developing onsite genotyping of Antarctic penguins

This research profile highlights the increasing need for mobile qPCR testing using an example from conservation biology. Read how PrimeTime® Custom qPCR Assays have been designed to distinguish genetic variants of Adélie penguins.

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Strategies for optimizing high throughput qPCR for expression profiling

Webinar summary: Learn how to address the challenges of high throughput RT-qPCR expression profiling from a prominent qPCR expert, Dr. Mikael Kubista (TATAA Biocenter, Sweden).

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Could your PCR be affected by contamination?

Learn how to prevent false amplification from DNA contamination, and how to address contamination when it does occur.

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Developing a qPCR point-of-care diagnostic for Ebola

Learn how Ubiquitome, Battelle, and IDT are developing a rapid qPCR Ebola virus test for easy use in the field. The PrimeTime qPCR assay is designed to be run on Ubiquitome’s hand-held, battery powered real-time PCR device, the Freedom4.

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Decrease qPCR background, improve qPCR signal

Product spotlight: See data demonstrating that probes containing a second, internal quencher provide increased signal detection and greater assay sensitivity in qPCR assays vs. single-quenched probes, such as probes with BHQ Quenchers. ZEN™ and TAO™ Double-Quenched Probes also make it more feasible to use longer probes due to the resulting lower background fluorescence.

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Choosing a qPCR assay: Inventoried or predesigned?

You may know that several companies offer inventoried qPCR assays. But did you know that these assays are often pre-manufactured and stocked, waiting perhaps for years for that order to be placed? In contrast, IDT PrimeTime® Predesigned qPCR Assays are manufactured at the time of order, and can therefore take into account sequence updates such new annotations.

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Design efficient PCR and qPCR primers and probes using online tools

Simplify planning of your qPCR experiments using IDT free, online tools for oligonucleotide analysis and PCR primer design. This article provides an overview of our predesigned qPCR assays and the basics of designing customized PCR primers and hydrolysis probes with the PrimerQuest® Tool.

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Do your qPCR assays come with sequence information? They should. Here Is why.

qPCR assays (primer & probe sets) from other suppliers are often provided without sequence information. IDT always gives you the sequences to the oligos you order. And that can be very important. Read why.

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PrimerQuest® Tool: From basic to highly customizable designs

Read these tips on how to use the PrimerQuest Tool to customize primers and probe for a wide variety of qPCR applications. Examples show how to adjust reaction conditions, add a probe to a set of previously designed primers, define primer positions, and include or exclude sequences from the assay designs.

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Selecting qPCR assays in plates for high throughput and large-scale studies

Learn some tricks for easy ordering of qPCR assays in plates.

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Multiplex qPCR—how to get started

Learn how multiplex qPCR can save sample, reagent cost, and time. The article provides recommendations for multiplex qPCR assay design and experimental setup.

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qPCR with intercalating dyes: PrimeTime® qPCR Primers

Need qPCR assays to use with intercalating dyes? You can obtain PrimeTime qPCR Primers to detect genes in human, mouse, and rat transcriptomes with intercalating dyes such as SYBR® Green and EvaGreen®. Because our primer only and probe-based assays use the same designs, when you decide to include probes in your assays, you can just request the probes that go along with these same primer sequences.

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Dengue, Zika transmission slowed by Wolbachia bacterium

The Eliminate Dengue Program is a multinational research organization dedicated to reducing the spread of mosquito-transmitted diseases. Learn about how IDT is supporting their work, which has recently made breakthroughs for dengue and Zika virus mitigation.

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Use splice junctions to your advantage in qPCR

Get recommendations for avoiding PCR amplification of genomic DNA, as well as for identifying and quantifying splice variants, in this article on designing qPCR assays that span splice junctions.

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A qPCR master mix stable enough for ambient shipping

Product spotlight: Choose a qPCR master mix shipped at ambient temperature. Why? PrimeTime® Gene Expression Master Mix consistently provides high qPCR efficiency, and ambient shipping directly benefits you by reducing shipping costs and time. Ambient shipping is also environmentally friendlier, as fewer resources are required to make and dispose of dry or gel ice packaging materials. Learn more about the strong thermal stability of this master mix.

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Zika virus: Advances in disease modeling and detection

IDT is supporting global research aimed at reducing the widespread effects of Zika. Learn about the virus, and read a summary of the latest developments.

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qPCR assays for optimized viral detection in clinical samples

Learn how ZEN™ Double-Quenched Probes were used in a unique qPCR experiment to help rapidly and accurately detect the highly variable norovirus in clinical samples.

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Increase the Tm of short, AT-rich primers and probes

Modification highlight: Add this modified base to increase the melting temperature (Tm) of primers and probes. It is especially useful when you need to work with short A-T rich sequences.

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Obtain high efficiency qPCR results using PrimeTime® Gene Expression Master Mix

Product Spotlight: If you are doing 5′ nuclease assays for qPCR or 2-step qPCR, use this versatile master mix in both singleplex or multiplex. This master mix is compatible with a wide range of instruments—whether you prefer standard or fast cycling conditions, and whether or not you need a reference dye.

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Increase sensitivity and precision in your qPCR experiments

Learn how you can use double-quenched probes to decrease background, and increase sensitivity and precision in your qPCR experiments.

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DRONE delivers oligos for promoter oligonucleotide pull-down assay

An IDT customer who is using IDT oligos for a transcription factor pull-down assay and qPCR assays for expression analysis, wins an April Fools' Day drone prize.

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MGB Eclipse® Probes for human in vitro diagnostics

Product spotlight: Obtain MGB Eclipse® Probes for human in vitro diagnostic end-use applications. Selecting MGB Eclipse® Probes made by the IDT GMP manufacturing division provides you with complete process transparency and product traceability, in addition to consistent, reliable oligonucleotide quality.

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She Prefers Red—Guppy Mate Preference

Scientists from Simon Fraser University (British Columbia, Canada) use guppies as a model to study the evolution of female mate preferences for male coloration. Read more about their field study using PrimeTime® qPCR Assays, ZEN™ Double-Quenched Probes, and gBlocks® Gene Fragments.

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Melt curve analysis for improved intercalating dye qPCR

Listen to this discussion of the benefits and limitations of melt-curve analysis using clear example—presented as a webinar. Read how PrimeTime Predesigned qPCR Primer Assays are designed to be compatible with intercalating dyes, and can also be transitioned to probe-based assays.

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qPCR Probes—selecting the best reporter dye and quencher

Read these recommendations for choosing dyes and quenchers, taking into account instrument compatibility and multiplex probe applications.

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DNA mutations and copy number alterations identified in FFPE tumor samples suggest potential therapeutic targets

Citation summary: Next generation sequencing was used to identify DNA mutations and copy number alterations in FFPE phyllodes tumor samples. Results were validated with Sanger sequencing and IDT PrimeTime® qPCR Assays.

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Improving immuno-PCR by optimizing antibody-oligo conjugation

Immuno-PCR, a modified antigen detection method, uses antibodies coupled to DNA, followed by real-time PCR. Using IDT amino-modified oligos, see how this method can increase the sensitivity of antibody-target detection by 100–10,000X over standard ELISA assays. An Innova Biosciences kit and service include IDT oligos to generate the antibody-oligonucleotide conjugates. Innova affirms that the high quality of IDT oligos provides optimal conjugation.

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Validating sequencing results of a repetitive element with digital droplet PCR

Citation summary: Mobilization of long interspersed element 1 (L1) can be involved in human disease and cancer. See how the specificity and sensitivity of digital droplet PCR can be used to validate the detection of rare L1 insertion events.

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Discriminating highly similar transcripts using rhPCR

Research profile: Read about the uses of RNase H-dependent PCR, a technology developed by IDT to increase PCR specificity and eliminate unwanted interactions between primer sets (e.g., primer-dimers, etc.). An example is provided in which it is used to distinguishing highly similar alternatively spliced sequences. This technology can also be useful in genotyping applications, in highly multiplexed qPCR assays, library construction for Next Generation DNA Sequencing, and for rare allele detection, where the added specificity provided by the blocked-cleavable primers enables detection of a rare mutant allele in a background of large amounts of wild type DNA.

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Interpreting melt curves: An indicator, not a diagnosis

Performing intercalating dye PCR/qPCR assays? Review examples of PCR melt curve data with our scientists to determine what it can/cannot tell us about resulting PCR amplicons.

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Double-quenched probes increase sensitivity of qPCR assay detecting viral load

Citation summary: Use of a ZEN™ Double-Quenched Probe results in a marked decrease in background fluorescence compared to an identical TaqMan® probe containing only a single quencher. The data suggest that such double-quenched probes may be a better approach for other qPCR probe-based assays.

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Recommended dye combinations for multiplex qPCR

Recommendations for selecting dyes for multiplex qPCR that minimize background and avoid overlap of fluorescent signals. Included is a table of compatible dyes for multiplexing on common qPCR instruments and a list of suggested quenchers.

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Epigenetic biomarkers for prostate cancer

Research profile: See how these scientists use methylation and expression analysis methods to evaluate epigenetic markers for early, noninvasive detection of aggressive prostate cancer. IDT PrimeTime® qPCR Assays, ZEN™ Double-Quenched Probes, and gBlocks® Gene Fragments facilitate this research.

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Digital PCR (dPCR)—What is it and why use it?

Read this general summary of dPCR, and how it can be used for qPCR applications, including multiplex qPCR.

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Understanding melting temperature (Tm)

Read this advice from our own thermodynamics specialist, Dr Richard Owczarzy, on the effects of melting temperature (Tm) on hybridization. He provides considerations for better oligo and PCR/qPCR assay design, including oligo concentration, salt, and base pairing mismatch positioning.

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A fast, sensitive, cost-effective alternative to radiolabeling and HPLC/MS for measuring dNTPs

Citation summary: A rapid and sensitive fluorescence-based method that uses synthetic templates (IDT oligonucleotides), a PrimeTime® Primer, and ZEN™ Double-Quenched Probes for quantifying cellular dNTPs.

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Designing PCR primers and probes

Many factors can influence successful PCR experiments, including primer and probe location, length, interaction and self-folding, melting temperature, annealing temperature, and GC content. Review these general recommendations for designing primers and probes and for choosing target locations for PCR amplification.

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qPCR probes—get the right scale at the right price

Only running 100–500 amplification reactions with FAM-labeled probes? Get a better price by using the PrimeTime Eco probes mid-range reaction scale for researchers. It's cheaper per reaction than larger reaction sizes and provides double-quenched FAM probes.

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Exon numbering—not as easy as 1, 2, 3...

Exon numbering and location data can differ across various software tools, including with NCBI's gene database. For example, exons within alternatively spliced transcripts are sometimes individually numbered, with no consistent gene-based numbering system across these transcripts for identifying exons. Learn how IDT exon location information gives each exon a unique number and how that compares with the NCBI naming system.

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Optimizing multiplex qPCR for detecting infectious diseases and biothreat agents in the field

Researchers at Tetracore specialize in developing large sets of robust probe-based qPCR assays for use in a multiplex format to detect infectious diseases and bio-terrorism threat agents. Here they discuss the need to: use probe dyes compatible on common PCR instruments, maintain low background with multiple probes, and reformulate assays to address viral mutation; and how ZEN™ Double-Quenched Probes have helped meet these criteria.

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Double-quenched probes increase qPCR sensitivity and precision

Data demonstrating that ZEN™ Double-Quenched Probes in qPCR assays provide increased signal detection and greater assay sensitivity than single-quenched probes, such as probes with BHQ Quenchers. ZEN™ Double-Quenched Probes also make it more feasible to use longer probes due to the resulting lower background fluorescence.

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GAPDH, a good reference sequence?

What internal controls are you using? Housekeeping genes are frequently used as internal controls. However, they may not be the best choices due to lesser known functions and the presence of pseudogenes, some of which, as in the case of GAPDH—used here as an example—may be expressed.

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Easily designed standard curves for qPCR

Adopt this easy way to combine control templates/multiple targets onto a single construct, and get the advantages that they provide for PCR experiments.

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Digital PCR—Simplifying quantitative PCR

Review the theory behind why digital PCR makes gene expression quantitation easier & more accurate. Raindance Technologies describes how they use IDT ZEN & LNA probes to improve the signal-to-noise ratio and rare allele detection in their multiplex experiments.

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How to avoid false positives in PCR and what to do if you get them

Do you know what causes false positives in the Negative Template Control sample during PCR? Review the causes and get these suggestions for preventing them.

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DNA methylation analysis—keeping it simple

Research profile: Use of methylation-sensitive restriction enzymes and probe-based PCR to provide methylation percentage for specific amplicon regions (primarily promoters). Read how IDT ZEN Double-Quenched Probes, PrimeTime qPCR Assays, and gBlocks Gene Fragments augment this analysis.

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Predictable control of gene expression by mRNA, 3’ untranslated region motifs

Citation summary: See how these researchers use gBlocks® Gene Fragments as qPCR standards to generate DNA standard curves for absolute quantification of mRNA.

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qPCR assay plates for large and high throughput studies

Customize qPCR assay plates for probe-based or primer alone assays. You can mix different dye-quencher combinations and request replicate plates. And no need to fill every well.

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Better PCR probes: A second quencher lowers background, increasing signal detection

Modification Highlight: Add the ZEN Quencher as a second, internal quencher in qPCR 5’-nuclease assay probes to obtain greater overall dye quenching, lowering background, and increasing signal detection. When incorporated into oligonucleotides, it also serves to strengthen duplex formation and block exonuclease digestion, while remaining nontoxic to cells. Thus the ZEN Quencher can be useful in steric blocking antisense oligonucleotide applications.

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qPCR validation of microarray data using PrimeTime® qPCR Assays and ZEN™ Double-Quenched Probes

Citation summary: See this example of how researchers use of ZEN™ Double-Quenched Probes with PrimeTime® qPCR Assays for qRT-PCR experiments to validate microarray data.

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Alternative dyes for FISH, FRET, and qPCR

Use ATTO™ dye labeled oligonucleotides as alternatives in applications including fluorescence in situ hybridization (FISH), fluorescence resonance energy transfer (FRET), and dual-labeled probes used in qPCR experiments.

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qPCR terminology—what does it mean?

Review these definitions of some of the most commonly used terms and distinctions encountered in qPCR experiments.

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Barcoding life

Research profile: The International Barcode of Life Project involves the construction of a comprehensive database of DNA sequence tags for eukaryotic life that links genetic, morphological, and ecological data. High throughput batch processing of samples requires quality and consistency of all components. Learn how the researchers use cocktails of IDT primers that work efficiently across a variety of taxa to amplify target DNA fragments.

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A conversation about qPCR with Jo Vandesompele

Research profile: An interview with Dr Jo Vandesompele, internationally recognized expert on quantitative PCR, discussing the common issues researchers face when using qPCR, and the future direction of qPCR technology.

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The Story of Taq and Thomas Brock’s Thermophiles

Do you know the origin of Taq polymerase? Find out in this quick review.

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qPCR probes—achieve high Tm without sacrificing quenching

Need a longer probe, or one with a higher Tm? Placed directly between DNA bases, the ZEN Internal Quencher eliminates the need for LNAs, MGB, or internal quenchers attached to thymidine bases. When combined with a 3’ quencher, you can design longer probes with sufficient melting temperature for qPCR while maintaining a consistently low background.

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Sample preparation for successful qPCR

Read about qPCR sample preparation and what experimental details you should consider for obtaining accurate and consistent results.

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Steps for a successful qPCR experiment

Read these recommendations for 5′ nuclease assay design and experimental setup that will help you obtain accurate and consistent results.

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Starting with RNA—one‑step or two‑step RT‑qPCR?

Starting with RNA? When performing real-time qPCR, one has to decide whether to use a one-step protocol that combines the RT reaction and PCR in one tube, or a two-step protocol where the RT reaction is performed separately from the PCR. Here are some guidelines.

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Identifying stem cell differentiation biomarkers

Research profile: Scientists at ReGenesys are using PrimeTime qPCR Assays in multiplex qPCR experiments to identify biomarkers that allow them to stage cells as they de-differentiate into stem cells. Learn about their work with a bone marrow derived cell line, called MultiStem, a primitive mesenchymal stem cell variant. MultiStem cells have the potential to be used as an off-the-shelf product to treat several diseases.

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Using qPCR assays in multiplex experiments

Product spotlight: Starting multiplex qPCR experiments? IDT PrimeTime Predesigned and Custom qPCR Assays offer multiple dye/quencher combinations and primer/probe ratios to simplify the multiplex experiment design.

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Tips for using BLAST to locate PCR primers

Need a quick way to find the location of primers within a gene or the expected size of the resultant PCR product? In this tip we show you how to get this information using BLAST.

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SYBR® to 5' nuclease assays

Product spotlight: Researchers often use intercalating dyes, like SYBR® Green I, for qPCR experiments for the advantage they bring in lowered price and reduced turnaround time. However, the disadvantages to using these reagents are significant. Read how easy it is to convert to 5’ nuclease assays and the advantages the addition of a hydrolysis probe will provide.

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Predesigned qPCR Assays

Probe-based qPCR assays for quantification of human, mouse, and rat gene expression. Order in plates or tubes.

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Related Articles

Designing PCR Primers and Probes

General guidelines for designing primers and probes and for choosing target locations for PCR amplification.

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Steps for a Successful qPCR Experiment

Considerations for 5′ nuclease assay design and experimental setup to help you obtain accurate and consistent results.

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Interpreting Melt Curves: An Indicator, Not a Diagnosis

Examining PCR melt curve data to determine what it can/cannot tell us about resulting PCR amplicons.

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Epigenetic Biomarkers for Prostate Cancer

Scientists use methylation and expression analysis methods to evaluate epigenetic markers for early, noninvasive detection of aggressive prostate cancer. IDT PrimeTime® qPCR Assays, ZEN™ Double-Quenched Probes, and gBlocks® Gene Fragments facilitate this research.

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Optimizing Multiplex qPCR for Detecting Infectious Diseases and Biothreat Agents in the Field—ZEN™ Double-Quenched Probes bring down the background

Tetracore researchers developing large sets of robust probe-based qPCR assays discuss the need to: use probe dyes compatible on common PCR instruments, maintain low background with multiple probes, and reformulate assays to address viral mutation.

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