Real-Time qPCR and RT-qPCR Kits

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
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

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

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

  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

  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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