Promega Corporation

Real-Time qPCR and RT-qPCR Kits

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  • Choosing a Kit
  • 1 or 2-Step?
  • Dye-Based qPCR
  • Probe-Based qPCR
  • Primer-Based qPCR

Promega Real-Time PCR Systems at a Glance

  Dye-Based Detection Probe-Based Detection Primer-Based Detection
GoTaq® Dye-Based
qPCR and RT-qPCR
GoTaq® Probe-Based
qPCR and RT-qPCR
Plexor® qPCR and RT-qPCR Systems
Technology Uses dsDNA binding dye such as BRYT Green™ Dye to detect PCR product as it accumulates during PCR. Uses hydrolysis probes specific to target gene to detect product as it accumulates during PCR. Uses modified labeled base pair incorporation to quenching the fluorescent signal during PCR.
Specificity Medium High High
Sensitivity-Low Copies Variable 1-10 copies 1-10 copies
Reproducibility Medium High High
Multiplexing checkMark checkMarkcheckMark checkMarkcheckMark
Melt-curve for specificity Yes No Yes
Cost $ $$$ $$
Applications Gene expression
DNA quantitation
CHiP
Gene expression
DNA quantitation
CHiP
SNP genotyping
Copy number variation
Pathway analysis
microRNA & small RNAs
Mutation Detection
Protein Analysis
Multiplexing
Gene expression
DNA quantitation
CHiP
SNP genotyping
Copy number variation
Pathway analysis
microRNA & small RNAs
Mutation Detection
Protein Analysis
Multiplexing
Ordering Information Ordering Information Ordering Information

Choosing Between 1-Step and 2-Step RT-qPCR Systems

Promega GoTaq® Probe and Dye Based RT-qPCR Kits are offered in 1-Step and 2-Step formats.
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
  • You will 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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Ultra-bright qPCR Master Mix for Higher Sensitivity and Earlier Detection

GoTaq® qPCR Master Mix contains a new DNA-binding dye that exhibits greater fluorescence than SYBR® Green. Combined with the GoTaq® Hot Start enzyme and an optimized buffer, GoTaq® qPCR Master Mix provides robust real-time PCR with increased reliability, reproducibility and sensitivity.

  • Higher fluorescence, often resulting in earlier Cqs
  • Direct substitute for SYBR® Green I products
  • Enhanced stability for automated setup
  • Choose from qPCR, 1-Step or 2-Step RT-qPCR options
  • Compatible with both fast and standard cycling methods
  • Sensitive quantitation on most real-time instruments
Ordering Information
See Data from GoTaq Users
 8275TA GoTaq® qPCR Master Mix provides high sensitivity in detecting a single-copy gene.

GoTaq® qPCR Master Mix provides high sensitivity in detecting a single-copy gene.

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Sensitive, Resistant to Inhibitors, Ready-to-Use Master Mixes for Probe-Based qPCR

The GoTaq® Probe qPCR and RT-qPCR Systems are ready-to-use 2X master mixes optimized for quantitative PCR in the hydrolysis probe detection format. The 1-Step and 2-Step RT-qPCR Master Mixes contain GoScript™ Reverse Transcriptase for efficient first-strand cDNA synthesis. The GoTaq® Probe 1-Step RT-qPCR System is formulated with dUTP. When dUTP is incorporated into the amplification products, the amplicons are susceptible to degradation by uracil-DNA glycosylase (UNG); this allows users to incorporate UNG into subsequent reactions for control of possible carryover contamination.

  • Resistant to inhibitors
  • Easy room-temperature setup
  • Choose from qPCR, 1-Step or 2-Step RT-qPCR options
  • Compatible with both fast and standard cycling methods
  • Sensitive quantitation on any real-time instrument
Ordering Information
 11148TA GoTaq® Probe 2-Step RT-qPCR System cDNA was generated from 100ng of human pancreas total RNA, using the GoScript™ Reverse Transcription System. The human insulin gene (INS) was detected from five-fold serial dilutions of cDNA (100ng to 6.4pg), using GoTaq® Probe qPCR Master Mix for amplification. The resulting standard curve is shown in the inset (R2 = 0.998, slope = –3.33).

cDNA was generated from 100ng of human pancreas total RNA, using the GoScript™ Reverse Transcription System. The human insulin gene (INS) was detected from five-fold serial dilutions of cDNA (100ng to 6.4pg), using GoTaq® Probe qPCR Master Mix for amplification. The resulting standard curve is shown in the inset (R2 = 0.998, slope = –3.33).

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Multiplexing Made Easy 

The Plexor® qPCR and qRT-PCR Systems are multiplex-capable real-time amplification systems based on a novel base pair chemistry. Each target is measured directly during amplification and not through a secondary reaction. Plexor® reactions require only two primers for each target. One primer contains both a fluorescent tag and modified base. As amplification proceeds, fluorescence is reduced by site-specific incorporation of a quencher opposite the complementary modified base.

  • Only two primers are required for each target, making the design of multiplex assays much easier
  • Measure controls and targets at the same time in the same assay well
  • Melt curve analysis for specificity confirmation
  • Choose from qPCR, 1-Step and 2-Step RT-qPCR options 
  • Compatible with most real-time instruments capable of measuring more than one fluor
  • The Plexor® Analysis Software allows users to import and analyze data from their preferred instrument platform
Ordering Information
Primer Design & Instrument Support
 5756TA Amplification and standard curves. Panel A. A representative amplification curve, which shows the relative fluorescence units (RFU) at each cycle of the reaction. The amplification threshold is indicated by a horizontal line across the graph. This threshold is used to establish the cycle threshold (Ct), the cycle at which the amplification curve crosses the amplification threshold, for each sample. Panel B. A standard curve generated from the amplification curve data shown in Panel A. Data were collected on an Applied Biosystems 7500 Real-Time PCR System and analyzed using the Plexor™ Analysis Software.

Amplification and standard curves. Panel A. A representative amplification curve, which shows the relative fluorescence units (RFU) at each cycle of the reaction. The amplification threshold is indicated by a horizontal line across the graph. This threshold is used to establish the cycle threshold (Ct), the cycle at which the amplification curve crosses the amplification threshold, for each sample. Panel B. A standard curve generated from the amplification curve data shown in Panel A. Data were collected on an Applied Biosystems 7500 Real-Time PCR System and analyzed using the Plexor™ Analysis Software.

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