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Two-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-reviewed studies. Explore below to see how we can solve your challenges with two-step reverse transcription quantitative PCR (RT-qPCR).

Advantages of two-step RT-qPCR

  • Compatible with limited sample input due to high sensitivity
  • Able to optimize RT and qPCR steps separately
  • Can generate cDNA stocks

Limitations

  • More time-consuming
  • Less amenable to high-throughput workflows

Introduction  

Quantitative PCR (qPCR) is a common, powerful technique for the accurate analysis of gene expression. When starting with RNA samples, you must first perform a reverse transcription (RT) step to generate cDNA for the subsequent qPCR reaction. Two-step RT-qPCR performs the RT step in one tube and the qPCR reaction in a separate tube (Figure 1).

Two-step RT-qPCR

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

Two-step RT-qPCR employs a two-tube workflow that makes it possible to optimize the RT and qPCR steps separately, allowing maximum flexibility, specificity, and efficiency. Two-step RT-qPCR works best with workflows analyzing many targets in fewer samples. Another advantage is that two-step RT-qPCR generates cDNA in a separate tube from the qPCR reaction, allowing for cDNA stocks to be banked for future use.

Two-step RT-qPCR has some limitations. In general, the protocols are more time-consuming and involve more pipetting steps. This introduces more variability and increases the risk of contamination. Additionally, while the ability to optimize both the RT and qPCR steps separately can improve sensitivity and efficiency, it comes with the drawback of having to optimize two reactions instead of one. Lastly, the two-tube protocol cannot be as easily adapted to automated, high-throughput workflows, necessitating more hands-on-time.

We offer two-step RT-qPCR kits in multiple formats for convenience, flexibility, and specificity. All kits can be combined with our probe- or TB Green- (our proprietary green intercalating dye) based qPCR kits to meet your experimental needs and give you the flexibility to run a wide variety of applications.

Two-step RT-qPCR using PrimeScript RT  

Our two-step RT-qPCR kits are optimized for real-time PCR. Each contains PrimeScript Reverse Transcriptase, which has exceptionally strong strand displacement activity for optimal efficiency during cDNA synthesis. The PrimeScript reverse transcriptase is robust, versatile, and well-suited for applications requiring full-length cDNA synthesis. It enables reverse transcription of virtually any RNA template, including templates that are GC-rich and/or have high levels of secondary structure, making our two-step RT-qPCR kits ideal for both routine and challenging RT applications. PrimeScript RTase is a modified, recombinant MMLV (Moloney Murine Leukemia Virus) enzyme, verified to be RNase H Minus, and is engineered to allow full-length cDNA synthesis reactions at lower temperatures (e.g., 42°C). This decreases the risk of RNA degradation that occurs during conventional reactions performed at higher temperatures.

We provide our two-step RT-qPCR kits in three formats to suit your experimental needs:

Highlighted citations  

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

Makino, K. et al. Inhibition of uterine sarcoma cell growth through suppression of endogenous tyrosine kinase B signaling. PLoS One 7, e41049 (2012).

Cat. No. RR037A was used to analyze gene expression levels in frozen and FFPE uterine leiomyosarcoma samples. The findings implicated TrkB signaling and provided potential drug targets for cancer therapies.

Mizutani, R. et al. Identification and characterization of novel genotoxic stress-inducible nuclear long noncoding RNAs in mammalian cells. PLoS One 7, e34949 (2012).

Cat. No. RR036A was used to identify a variety of novel lncRNAs using transcriptomic sequencing and bioinformatics.

Sato, Y., Inoue, M., Yoshizawa, T. & Yamagata, K. Moderate hypoxia induces β-cell dysfunction with HIF-1-independent gene expression changes. PLoS One 9, e114868 (2014).

Cat. No. RR047A was used to detect alterations of gene expression in primary islet cells under hypoxic conditions. These findings could implicate novel roles in β-cell dysfunction in type 2 diabetes.

 

Featured Products

Cat. # Product Size License Quantity Details
RR036A PrimeScript™ RT Master Mix (Perfect Real Time) 200 Rxns USD $599.00

This product is a reverse transcription reagent kit designed to perform reverse transcription optimized for two step real-time RT-PCR (RT-qPCR). It contains a 5X pre-mixed reagent containing all of the components needed for quantitative RT-PCR reverse transcription (PrimeScript RTase, RNase Inhibitor, Random 6 mers, Oligo dT Primer, dNTP Mixture, and reaction buffer), and a reaction can be started simply by adding template RNA and water. Because it uses PrimeScript RTase, which features excellent extension, this product makes it possible to synthesize template cDNA for real-time PCR efficiently in a short time. The cDNA obtained with this product can be used in both intercalating green dye qPCR assays and probe qPCR assays.

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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RR036B PrimeScript™ RT Master Mix (Perfect Real Time) 800 Rxns USD $1918.00

This product is a reverse transcription reagent kit designed to perform reverse transcription optimized for two step real-time RT-PCR (RT-qPCR). It contains a 5X pre-mixed reagent containing all of the components needed for quantitative RT-PCR reverse transcription (PrimeScript RTase, RNase Inhibitor, Random 6 mers, Oligo dT Primer, dNTP Mixture, and reaction buffer), and a reaction can be started simply by adding template RNA and water. Because it uses PrimeScript RTase, which features excellent extension, this product makes it possible to synthesize template cDNA for real-time PCR efficiently in a short time. The cDNA obtained with this product can be used in both intercalating green dye qPCR assays and probe qPCR assays. Cat. # RR036B contains 4 of Cat. # RR036A. Please refer to Cat. # RR036A 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.

Documents You May Also Like Image Data
RR037A PrimeScript™ RT Reagent Kit (Perfect Real Time) 200 Rxns USD $399.00

PrimeScript RT Reagent Kit is designed to perform reverse transcription optimized for real-time RT-PCR. It uses PrimeScript Reverse Transcriptase, which features excellent extension. The kit makes fast, efficient cDNA template synthesis for real-time PCR possible. This kit's protocol is simple and suitable for high throughput analysis. It can be used in combination with real-time PCR reagents such as TB Green Premix Ex Taq II (Tli RNaseH Plus) (Cat. #RR820A/B), TB Green Fast qPCR Mix (Cat. #RR430A/B), TB Green Premix Ex Taq (Tli RNaseH Pluse) (Cat. #RR420A/B), or Probe qPCR Mix (Cat. #RR391A/B) for two step real-time RT-PCR. The optimized protocol for assay can be selected in each assay condition using either TB Green or a probe.

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.

Documents Components Image Data Resources
RR037B PrimeScript™ RT Reagent Kit (Perfect Real Time) 800 Rxns USD $1267.00

PrimeScript RT Reagent Kit is designed to perform reverse transcription optimized for real-time RT-PCR. It uses PrimeScript Reverse Transcriptase, which features excellent extension. The kit makes fast, efficient cDNA template synthesis for real-time PCR possible. This kit's protocol is simple and suitable for high throughput analysis. It can be used in combination with real-time PCR reagents such as TB Green Premix Ex Taq II (Tli RNaseH Plus) (Cat. #RR820A/B), TB Green Fast qPCR Mix (Cat. #RR430A/B), TB Green Premix Ex Taq (Tli RNaseH Pluse) (Cat. #RR420A/B), or Probe qPCR Mix (Cat. #RR391A/B), for two step real-time RT-PCR. The optimized protocol for assay can be selected in each assay condition using either TB Green or a probe. Cat. # RR037B contains 4 of Cat. # RR037A. Please refer to Cat. # RR037A 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.

Documents Image Data
RR047A PrimeScript™ RT Reagent Kit with gDNA Eraser (Perfect Real Time) 100 Rxns USD $599.00

PrimeScript RT reagent Kit with gDNA Eraser is a reverse-transcription kit for real-time RT-PCR (RT qPCR) that includes a genomic DNA elimination reaction. cDNA synthesis from RNA can be achieved without sample loss in a rapid reaction that is complete in less than 20 minutes. Genomic DNA is eliminated by treatment for 2 minutes at 42℃ with gDNA Eraser, which has potent DNA degradation activity. Then a reverse-transcription reaction reagent is added that includes a component which completely inhibits DNA degradation activity, and the reverse-transcription reaction proceeds for 15 minutes. The cDNA obtained using this product can be used with either an intercalating green dye qPCR assay or probe qPCR assay. Please use in combination with quantitative PCR reagents such as TB Green Premix Ex Taq II (Tli RNaseH Plus) (Cat. #RR820A) and Probe qPCR Mix (Cat. #RR391A).

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.

Documents Components You May Also Like Image Data Resources
RR047B PrimeScript™ RT Reagent Kit with gDNA Eraser (Perfect Real Time) 400 Rxns USD $2031.00

PrimeScript RT reagent Kit with gDNA Eraser is a reverse-transcription kit for real-time RT-PCR (RT qPCR) that includes a genomic DNA elimination reaction. cDNA synthesis from RNA can be achieved without sample loss in a rapid reaction that is complete in less than 20 minutes. Genomic DNA is eliminated by treatment for 2 minutes at 42°C with gDNA Eraser, which has potent DNA degradation activity. Then a reverse-transcription reaction reagent is added that includes a component which completely inhibits DNA degradation activity, and the reverse-transcription reaction proceeds for 15 minutes. The cDNA obtained using this product can be used with either an intercalating green dye qPCR assay or probe qPCR assay. Please use in combination with quantitative PCR reagents such as TB Green Premix Ex Taq II (Tli RNaseH Plus) (Cat. #RR820A) and Probe qPCR Mix (Cat. #RR391A). Cat. # RR047B contains 4 of Cat. # RR047A. Please refer to Cat. # RR047A 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.

Documents You May Also Like Image Data

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.

Learn more Product details

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