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Real-Time qPCR Overview

A Long History of Innovative Research Supports Our Real-Time qPCR Reagents

Takara Clontech's extensive history of innovative research in genetics and PCR

Takara Clontech scientists are expert enzymologists who are continually working to improve enzymes and buffers for PCR, real-time qPCR, and RT-PCR.

You expect a lot from your real-time qPCR reagents—they need to perform at a high level and provide reproducibility, instrument compatibility, and sensitivity at a great value. Our real-time qPCR kits and master mixes are designed with these issues in mind. Whether your experiments are routine, complicated, or proving difficult, we have the right qPCR tools to move your research forward.

Learn more about the features of our qPCR reagents below.

High Assay Efficiency

High assay efficiency is critical to obtaining accurate qPCR data. Several factors can affect the overall efficiency of qPCR, including the reagent used for amplification. Our master mixes and kits provide excellent, consistent efficiency compared to other commercially available qPCR reagents.

High qPCR efficiency with SYBR Premix Ex Taq

PCR efficiency was assessed using SYBR® Premix Ex Taq (Tli RNase H Plus) and other commercially available SYBR master mixes (Company A, B, and C). Three portions of the ACTB gene were amplified (red: 186 bp; black: 381 bp; blue: 533 bp) from cDNA synthesized using 10 pg to 1 µg of human testes total RNA, and the standard curves were plotted.

Optimized Assay Specificity

Specificity is important—you don’t want to detect products that aren’t really there, and you don’t want your results to be artificially inflated by non-specific amplification products. Good primer design can significantly reduce this problem, but the formulation of your master mix is also important. The enzyme and buffer in each of our qPCR master mixes have been optimized to maximize specificity even for difficult amplifications, such as GC-rich templates.

Increased assay specificity with Takara Clontech SYBR reagents

Assay specificity was assessed using SYBR Premix Ex Taq II (Tli RNaseH Plus) and other commercially available SYBR master mixes (Company A and B). cDNA was synthesized from 10 pg to 1 µg of human testes total RNA. The expression of ACTB (186 bp and 381 bp fragments) was measured by real-time PCR using a StepOnePlus system (Life Technologies). Non-specific amplification was not observed for reactions with Takara Clontech’s premix.

qPCR master mixes optimized for specificity with GC-rich targets

Assay specificity for GC-rich targets was assessed using SYBR Premix Ex Taq II (Tli RNaseH Plus) and other commercially available SYBR master mixes (Company A and B). cDNA was synthesized from 10 pg to 1 µg of human testes total RNA using PrimeScript Reverse Transcriptase. The expression of C16orf84 (73% GC content) and PLSCR3 (67% GC content) was measured by real-time PCR using a StepOnePlus system (Life Technologies). The standard curves indicate that the SYBR master mix provided high-efficiency amplification. In addition, non-specific amplification was not observed for reactions with Takara Clontech’s premix, indicating high assay specificity for these GC-rich targets.

Versatile Instrument Compatibility

Our real-time qPCR master mixes have been tested for compatibility with a variety of instruments, and the corresponding user manuals include protocols for commonly used real-time PCR instruments. In addition to the instruments listed in the table below, researchers have successfully used SYBR Premix Ex Taq on the ABI 7900HT Fast Real-Time PCR System (Life Technologies), and SYBR Premix Ex Taq II on the CFX Connect Real-Time PCR Detection System (Bio-Rad).

qPCR Instrument Compatibility
  SYBR Premix Ex Taq (Tli Plus RNase H) SYBR Premix Ex Taq II (Tli Plus RNase H) Premix Ex Taq (Probe qPCR)
ABI PRISM 7000/7700 (Life Technologies)     Yes
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR Systems (Life Technologies) Yes Yes Yes
StepOnePlus Real-Time PCR System (Life Technologies) Yes Yes Yes
LightCycler (Roche) Yes Yes Yes
Smart Cycler System/Smart Cycler II System (Cepheid) Yes   Yes
CFX96 Real-Time PCR Detection System (Bio-Rad)   Yes  
Mx3000P (Agilent Technologies) Yes    

Quick Data from Sample to Readout

Whether you need a high-throughput workflow or just want fast results, our real-time qPCR master mixes can reduce your run times. For maximum reaction speed, the master mixes can be used with fast mode thermal cycling conditions.

High reaction efficiency with a variety of extension times

Premix Ex Taq (Probe qPCR) has the same reaction efficiency with extension times of 20, 25, and 30 seconds on an Applied Biosystems 7500 Fast Real-Time PCR System (Life Technologies).

Faster results with fast mode thermal cycling conditions

Premix Ex Taq (Probe qPCR) has the same reaction efficiency with extension times of 20 and 30 seconds on a LightCycler 480 System (Roche).

Economical Kits

Our real-time qPCR master mixes stretch your research dollars without sacrificing quality. Make the switch and you can save hundreds or even thousands of dollars annually. Compare the qPCR reagent you are currently using to the list prices below.

qPCR Reagent Price Comparison
Product Cat. # List
Price
# 50 µl Rxns Price/
50 µl Rxn
# 20 µl Rxns Price/
20 µl Rxn
SYBR Premix Ex Taq (Tli RNase H Plus) RR420A $290 200 $1.45 500 $0.58
RR420B $566 400 $1.42 1,000 $0.57
RR420W $1,159 1,000 $1.16 2,500 $0.46
SYBR Premix Ex Taq II (Tli RNase H Plus) RR820A $290 200 $1.45 500 $0.58
RR820B $566 400 $1.42 1,000 $0.57
RR820W $1,159 1,000 $1.16 2,500 $0.46
Premix Ex Taq (Probe qPCR) RR390A $233 200 $1.17 500 $0.47
RR390B $463 400 $1.16 1,000 $0.46
RR390W $929 1,000 $0.93 2,500 $0.37

SYBR Master Mix Options to Meet Your Needs

SYBR Premix Ex Taq (Tli+) provides the most versatility for real-time qPCR

  • Robust performance for most targets, resulting in high efficiency and low Ct values
  • A fast extension rate, allowing high-yield amplification (low Ct values) for a broad range of amplicon sizes, including long targets (up to ~533 bp)

If you have tried to improve reaction specificity by optimizing primer design, but are still not getting optimal performance, use SYBR Premix Ex Taq II (Tli+).

  • Superior specificity by suppressing non-specific amplification and primer-dimer formation
  • Accurate analysis of GC-rich targets
Differentiating SYBR Premixes
  SYBR Premix Ex Taq (Tli+) (Cat. # RR420) SYBR Premix Ex Taq II (Tli+) (Cat. # RR820) Terra qPCR Direct SYBR Premix (Cat. # 638319)
Key features

Efficiency

Versatility

Specificity

No template purification required

Experimental requirements

Use when performing standard qPCR reactions

Use when working with long amplicons (~550 bp)

Use when optimal primer design is difficult or impossible

Use when performing standard qPCR reactions

Use when highly specific amplification is required

Use when working with crude tissue extracts or dirty samples

Notes

Performs well where other enzymes show low yield

Ideal for:

  • Gene expression analyses
  • Genotyping with genomic DNA templates
  • High-throughput analyses
  • Fast reactions

Supresses non-specific amplification and the formation of primer dimers

Ideal for:

  • Gene expression analyses
  • Genotyping with genomic DNA templates
  • High-throughput analyses
  • Fast reactions

Great for high-throughput applications and GC-rich targets (<70%)

Includes 2X master mix with SYBR Green I

High Yields of cDNA in only 15 Minutes

Conversion of mRNA to cDNA can introduce variability—reverse transcriptases (RTs) have different efficiencies that can be target-dependent. Secondary and tertiary RNA structures, variation in priming efficiency, and properties of the reverse transcriptase itself can affect cDNA synthesis. PrimeScript Reverse Transcriptase is a robust enzyme that can quickly (~15 minute reaction time) synthesize high yields of cDNA even from the most difficult RNA templates. The PrimeScript RT kits below generate cDNA that can be used directly in real-time qPCR.

If you want to… Then select 
Make cDNA for use in real-time PCR using a master mix of RT and primers PrimeScript RT Master Mix (Perfect Real Time)
Make cDNA for use in real-time PCR using a kit that includes separate RT and primers PrimeScript RT Reagent Kit (Perfect Real Time)
Make cDNA for use in real-time PCR using a kit that includes separate RT and primers, plus reagents for removal of genomic DNA PrimeScript RT Reagent Kit with gDNA Eraser for Perfect Real Time

Streamlined Detection of Gene Expression with One-Step RT-qPCR

Do you want a more streamlined workflow? In one-step real-time RT-qPCR, all reactions (cDNA synthesis, PCR amplification, and detection) are performed in a single tube. A one-step protocol is simple and convenient, and minimizes the possibility of reaction contamination. We provide one-step real-time RT-PCR kits for both SYBR Green and TaqMan® probe detection. These kits include PrimeScript RT, an RNase H Minus MMLV-based reverse transcriptase, TaKaRa Ex Taq HS, a high-efficiency hot start PCR enzyme, and a buffer system optimized for one-step RT-PCR.

One-step qPCR provides a convenient, streamlined workflow

The One Step SYBR PrimeScript RT-PCR Kit II was used to measure the level of HPRT1 and generate a standard curve. Human liver total RNA (6.4 pg–100 ng) was used as a template and reactions were run following the recommended protocol. Amplification curves were generated for all of the template concentrations. The overlapping melt curves indicate that the same amplification products were generated even when different amounts of template were used. The data generated a linear standard curve within the wide range of RNA template concentrations.

Real-Time qPCR Product List

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