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Product Overview

One-step RT-qPCR kits

At Takara Bio, great enzymes are part of our DNA. Thanks to over 90 years of experience in the pursuit of excellence in biochemistry and molecular biology technologies, we have developed an extensive portfolio of tools that have been widely published in peer-review studies. Explore below to see how we can solve your challenges with one-step reverse transcription quantitative PCR (RT-qPCR).

Advantages of one-step RT-qPCR

  • Simple and rapid workflow
  • Compatible with large numbers of samples (when looking at few target genes)
  • Adaptable to high-throughput/automated workflows

Limitations

  • Cannot optimize RT step
  • Does not generate stock cDNA
  • Not ideal when analyzing large numbers of target genes
Introduction Principle of one-step RT-qPCR Probe-based detection TB Green-based detection Highlighted citations

Introduction  

Quantitative PCR (qPCR) is a powerful technique for the accurate analysis of gene expression. When starting with RNA samples, one must first perform a reverse transcription (RT) step to generate cDNA for the subsequent qPCR reaction. One-step RT-qPCR streamlines this workflow by performing the RT step in the same tube as the qPCR reaction (Figure 1).

one-step RT-qPCR

Figure 1. Schematic of a one-step RT-qPCR reaction.

In general, one-step RT-qPCR is best suited for applications where speed and throughput are required. As the single-tube protocol is easy to set up and compatible with liquid handlers and/or automated systems it allows for less hands-on time, reduces pipetting errors, and minimizes contamination. One-step RT-qPCR is best applied to workflows comprised of many samples with few target genes.

However, one-step RT-qPCR has some limitations. Since both the RT and qPCR steps take place in the same tube the reaction conditions cannot be optimized separately, which can lead to lower yields and/or efficiency in either step. Another limitation is that all the generated cDNA is used up in the subsequent qPCR step, meaning that no stocks of cDNA can be banked for further validation or experimentation.

We offer one-step RT-qPCR kits supporting both TB Green- (our proprietary green intercalating dye) and probe-based chemistries to meet your experimental needs and give you the flexibility to run a wide variety of applications.

Principle of one-step RT-qPCR  

Takara Bio's One Step PrimeScript III RT-qPCR Kit allows cDNA synthesis from RNA using PrimeScript Reverse Transcriptase, followed by PCR amplification with Takara Ex Taq Hot Start Version in a single, uninterrupted procedure. PCR amplification products are detected and monitored in real time with either probe- or TB Green-based detection.

Figure 2. Workflow schematic using our PrimeScript One Step RT-PCR Kit Ver.2.

Probe-based detection  

One-step RT-qPCR kits that utilize probe-based detection must be accurate, specific and reproducible. Our One Step PrimeScript RT-PCR Kit (Perfect Real Time) passes these requirements with flying colors. This kit is available in both 100 and 500 reaction sizes.

Oligonucleotides modified with a fluorophore (e.g., FAM) at the 5' end and quencher (e.g., TAMRA) at the 3' end are included in the reaction. During the annealing step, the probe specifically hybridizes to the template DNA and the fluorophore's fluorescence is suppressed by the quencher. During the extension step, the 5'→3' exonuclease activity of Taq DNA polymerase degrades the probe hybridized to the template. This prevents quenching and allows fluorescence emission. The amount of amplified product can be monitored by measuring the fluorescence intensity.

Figure 3. Schematic detailing the detection and quantitation of probe-based RT-PCR fluorescence.

TB Green-based detection  

One-step RT-qPCR kits that utilize TB Green-based detection are rapid, efficient, and sensitive. When you want rapid and cost-effective results in a single-tube protocol, utilize our One-Step TB Green PrimeScript RT-PCR Kit II (Perfect Real Time). This kit is available in both 100 and 500 reaction sizes.

This method detects fluorescence produced during the amplification process by adding a DNA intercalating dye (TB Green) that fluoresces upon binding to double-stranded DNA. Following the synthesis and binding of TB Green to DNA synthesized during RT-PCR, the quantity of amplified DNA and the melting point of the resulting amplicon can be measured.

Figure 4. Schematic detailing the detection and quantitation of TB Green-based RT-PCR fluorescence.

Highlighted citations  

Here are a few examples of research that's been driven by our one-step RT-qPCR kits:

Ma, W. et al. Zika virus causes testis damage and leads to male infertility in mice. Cell 167, 1511–1524.e10 (2016).

Cat. # RR064A was used to sensitively detect Zika viral RNA levels from multiple tissues in Zika virus-infected mice. These data were able to discern higher viral levels in testis, associated with infertility.

Xie, Y., Wang, M., Xu, D., Li, R. & Zhou, G. Simultaneous detection and identification of four sugarcane viruses by one-step RT-PCR. J. Virol. Methods 162, 64–68 (2009).

Cat. # RR064A was used in the development of a one-step quadruplex RT-PCR method for detecting viruses in sugarcane. This rapid and sensitive technique greatly reduced cost and labor since multiple infections could be tested for in one sample.

Zhang, N. et al. Development of one-step SYBR Green real-time RT-PCR for quantifying bovine viral diarrhea virus type-1 and its comparison with conventional RT-PCR. Virol. J. 8, 374 (2011).

Cat. # RR086A was used to develop a specific, sensitive, and reproducible assay for bovine viral diarrhea virus, a surrogate model for hepatitis C virus. The assay was 10-fold more sensitive than conventional assays and showed no primer dimers or nonspecific products.

Zou, Q. et al. Use of Praziquantel as an adjuvant enhances protection and Tc-17 responses to killed H5N1 virus vaccine in mice. PLoS One 7, e34865 (2012).

Cat. # RR086A was used to measure H5N1 infection levels in mouse lung tissue. This enabled the testing of a novel adjuvant, PZQ, which was able to reduce virus loads and prolong survival.

 

Featured Products

Cat. # Product Size Price License Quantity Details
RR064A One Step PrimeScript™ RT-PCR Kit (Perfect Real Time) 100 Rxns USD $337.00

The One Step PrimeScript RT-PCR Kit (Perfect Real Time) is designed for one step, real-time reverse transcription PCR (RT-PCR) using probe detection. With this kit, all RT-PCR steps can be performed in a single tube. Therefore, it is not necessary to add additional reagents during the reaction, minimizing the risk of contamination and simplifying the workflow. Amplified products are monitored in real time without need for gel electrophoresis after PCR. This kit is suitable for detection of small amounts of RNA. This kit uses PrimeScript Reverse Transcriptase, which allows efficient and rapid cDNA synthesis, and Takara Ex Taq HS, which enables highly efficient hot start PCR.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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RR064A: One Step PrimeScript RT-PCR Kit (Perfect Real Time)

RR064A: One Step PrimeScript RT-PCR Kit (Perfect Real Time)
RR064B One Step PrimeScript™ RT-PCR Kit (Perfect Real Time) 500 Rxns USD $1336.00

The One Step PrimeScript RT-PCR Kit (Perfect Real Time) is designed for one step, real-time reverse transcription PCR (RT-PCR) using probe detection. With this kit, all RT-PCR steps can be performed in a single tube. Therefore, it is not necessary to add additional reagents during the reaction, minimizing the risk of contamination and simplifying the workflow. Amplified products are monitored in real time without need for gel electrophoresis after PCR. This kit is suitable for detection of small amounts of RNA. This kit uses PrimeScript Reverse Transcriptase, which allows efficient and rapid cDNA synthesis, and Takara Ex Taq HS, which enables highly efficient hot start PCR. Cat. # RR064B contains 5 of Cat. # RR064A. Please refer to Cat. # RR064A for complete product documentation and resources.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

RR086A One-Step TB Green® PrimeScript™ RT-PCR Kit II (Perfect Real Time) 100 Rxns USD $337.00

One-Step TB Green PrimeScript RT-PCR Kit II (Perfect Real Time) is designed for intercalator-based, one-step real-time RT-PCR using TB Green. All steps of RT-PCR can be performed in a single tube, allowing a simple procedure and minimizing the risk of contamination. Also, amplified products are monitored in real time, so there is no need to verify by electrophoresis after PCR. This kit is suitable for detection of tiny amounts of RNA such as RNA viruses. This kit uses PrimeScript RTase, which has excellent extendibility and can efficiently synthesize cDNA quickly, and Takara Ex Taq HS, a high-efficiency hot start PCR enzyme, optimized for one-step RT-PCR. This kit achieves a simple and convenient procedure with premixed components.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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RR086A: One-Step TB Green PrimeScript RT-PCR Kit II (Perfect Real Time)

RR086A: One-Step TB Green PrimeScript RT-PCR Kit II (Perfect Real Time)
RR086B One-Step TB Green® PrimeScript™ RT-PCR Kit II (Perfect Real Time) 500 Rxns USD $1336.00

One-Step TB Green PrimeScript RT-PCR Kit II (Perfect Real Time) is designed for intercalator-based, one-step real-time RT-PCR using TB Green. All steps of RT-PCR can be performed in a single tube, allowing a simple procedure and minimizing the risk of contamination. Also, amplified products are monitored in real time, so there is no need to verify by electrophoresis after PCR. This kit is suitable for detection of tiny amounts of RNA such as RNA viruses. This kit uses PrimeScript RTase, which has excellent extendibility and can efficiently synthesize cDNA quickly, and Takara Ex Taq HS, a high-efficiency hot start PCR enzyme, optimized for one-step RT-PCR. This kit achieves a simple and convenient procedure with premixed components. Cat. # RR086B contains 5 of Cat. # RR086A. Please refer to Cat. # RR086A for complete product documentation and resources.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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Screening for novel coronavirus with one-step RT-qPCR: getting ahead of the outbreak

Learn how researchers identified a novel, highly contagious coronavirus and developed a one-step RT-qPCR screening protocol.

Read the blog article


SmartChip Real-Time PCR System

High-throughput real-time PCR

Where throughput meets flexibility

Real-time PCR (qPCR) is a powerful technique for genotyping and gene expression analysis. Currently, qPCR experiments are becoming increasingly complex—involving an expansive and growing list of targets from a larger number of samples, all with more technical replicates. The SmartChip Real-Time PCR System is a complete high-throughput solution that enables an unrivaled amount of flexible assay and sample formats, allowing researchers to seamlessly switch between dispensing assay reagents and samples into blank chips, or dispensing samples into custom, preprinted chips without the need for revalidation.

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