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qPCR and RT-qPCR Probe and Dye Systems

However the road curves, we're here to help you succeed

Real-time quantitative PCR, or qPCR, has become a standard tool in the analysis of nucleic acids. By incorporating fluorescently labeled probes or nucleic acids or fluorescent double-stranded DNA-binding dyes into the PCR reaction, product formation can be monitored following each PCR cycle. Since product formation is detected in the exponential phase of PCR where the reaction is most efficient, quantification of starting material is more accurate and sensitive.

Most common qPCR detection methods include probe-based as well as dye-based detection chemistries. Learn more about each type of method and the proven systems we offer.

Probe-Based PCR

Probe-based qPCR uses a fluorescent primer or probe to detect the amplification product. An advantage of using a labeled primer or probe is that it allows sequence-specific detection of the target, which reduces the likelihood of detecting nonspecific artifacts. Probe-based detection also can accommodate multiplex amplification, through the use of differentially labeled probes. There are a number of different approaches to probe-based qPCR detection including hydrolysis probes, hybridization probes, hairpin probes and labeled primers.

Promega's GoTaq® Real-Time PCR Systems provide hot-start activation, enhanced stability, and sensitive detection on most real-time PCR instruments, together with the convenience of a master mix formulation. With these Systems you can:

Achieve Superior Performance: sensitive detection for earlier quantitation of low- and high-copy-number targets over a broad dynamic range.

Rely on a Robust System: resistant to a wide range of PCR inhibitors.

Benefit from Versatility: amenable to both fast and standard cycling methods on most real-time PCR instruments using TaqMan® and other probe assays such as molecular beacons.

Run Multiplex Assays: buffers are compatible with the development of multiplexes

Appreciate Enhanced Stability: exceptional room-temperature setup is easy for automated and high-throughput detection.

Dye-Based PCR

The use of fluorescent DNA-binding dyes, like BRYT™ Green dye, is one of the most straightforward qPCR approaches used by scientists. A dye is added to the reaction, and fluorescence is measured at each PCR cycle. Because fluorescence of these dyes increases dramatically in the presence of double-stranded DNA, DNA synthesis can be monitored as an increase in fluorescent signal.

Thermal melt curves are generated by allowing all product to form double-stranded DNA at a lower temperature (approximately 60°C) and slowly ramping the temperature to denaturing levels (approximately 95°C). The product length and sequence affect melting temperature (Tm), so the melt curve is used to characterize amplicon homogeneity. By distinguishing between specific and nonspecific amplification products, the melt curve adds a quality control aspect during routine use. The generation of melt curves is not possible with assays that rely on the 5′→3′ exonuclease activity of Taq DNA polymerase, such as the probe-based TaqMan® technology.

The GoTaq® Dye-based Real-Time PCR Systems are optimized 2X master mixes for fast and reproducible quantitative PCR assays. They rely on BRYT Green® dye with minimal PCR inhibition for maximum PCR efficiency and greater fluorescence enhancement upon binding to double-stranded DNA. With these Systems you can:

Achieve Superior Performance: sensitive detection for reproducible and earlier quantitation of low- and high-copy-number targets over a broad dynamic range.

Rely on a Robust System: resistant to a wide range of PCR inhibitors. Benefit from Versatility: compatible with both fast and standard cycling methods on most real-time PCR instruments and typically delivers results in <50 minutes.

Appreciate Enhanced Stability: exceptional room-temperature setup is easy for automated and high-throughput detection.

qPCR Data Viewer

We have generated experimental data using different systems, instruments, templates, samples and more. Just select the options below to narrow the selection.

 
 

Helpful Resources

Real-time PCR (qPCR) is the workhorse of most gene expression studies. However, it can be overwhelming to a new user given the requirement for advanced instruments, vast choices for reagents, and the complexity of the technique. Here we provide an overview of basic qPCR principles, instruments, reagent considerations and common terms used in assay design and analysis.

Lab Chat

App Notes

Real-Time PCR Webinars

Introduction to Real-Time PCR: Basic Principles and Chemistries
Provides an overview of basic qPCR principles compared to traditional PCR and common terms used in assay design and analysis.

The Hows and Whys of Early Steps in RNA Analysis
Learn about the early steps of purification, protection from degradation and quantitation to improve your downstream analysis.

Optimize Your qPCR and RT-qPCR Assays with Careful Planning and Design
Covers multiple qPCR topics aimed at improving your qPCR results such as: optimizing qPCR assays including whether to use probe- or dye-based detection, choosing between 1-step and 2-step RT-qPCR assays, and overcoming key challenges such as sample integrity, multiplexing and the presence of inhibitors in the input sample.

Dealing with Reverse Transcription and Amplification Inhibitors: Reagent Choice Matters
Discusses the various types and sources of potential inhibitors and their potential impacts, with supporting data, and contrasts the performance of different downstream reagents in the presence of inhibitors.

Maximize Your Reverse Transcription-qPCR (RT-qPCR) Assays
Provides the basics of RT-qPCR including an overview of the technique, available options, data analysis, and interpretation. For the more experienced user includes in-depth coverage of common, but often misunderstood, problems and their solutions. Real-world examples of gene expression studies using this powerful technique are also presented.

Blogs

Video Protocol

Dye-based versus Probe-based Chemistries

  Dye-based qPCR Probe-based qPCR
Amplicon labeling dsDNA-binding dye Fluorescently labeled probes
Cost Lower cost Higher cost
Flexibility All optimized assays Single assay
Instrumentation All qPCR instruments Must match probes to filters
Specificity Measures all dsDNA Measures amplicon with probe sequence
Multiplexing No Yes –different dyes/filters
Melt analysis QC and genotyping Yes No
Throughput High Highest (multiplexed)
Sample required Low Lowest (multiplexed)
Requires validation Yes Yes

1-Step versus 2-Step qPCR Kits

Select 1-Step if: Select 2-Step if:
  • You do not need to store cDNA
  • You will dispose of samples after a few uses
  • You have many samples with one or few targets
  • You are using a liquid-handling robot
  • You need to store the cDNA
  • You have a limited amount of sample
  • You are assaying many targets per sample
1-Step Advantages 2-Step Advantages
  • Reduced chance of cross-contamination during the procedure
  • Can dispose of samples after a few uses
  • Faster results
  • Optimized performance of both RT and PCR steps
  • cDNA is available for other procedure
Disadvantages Disadvantages
  • Increased risk of primer-dimers
  • cDNA is not available for other assays
  • RT enzymes and buffers can inhibit real-time PCR
  • Less convenient, more time consuming
  • Contamination risk is higher than 1-step method

1-Step and 2-Step Protocol Overview

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