qPCR without optimization using TB Green Premix Ex Taq

Introduction

TB Green Premix Ex Taq (Tli RNase H Plus) master mix is optimized for efficiency and versatility, providing superior results when low yields or no amplification is obtained with other enzymes. In this experiment, Nox4 (NADPH Oxidase 4) mRNA expression was analyzed by qPCR using TB Green Premix Ex Taq master mix. Previous attempts to analyze this target using a SYBR green master mix from another supplier were unsuccessful. However, amplification with TB Green Premix Ex Taq master mix provided reliable results without any reaction optimization.

Results

Nox4 expression in THP-1 monocytes was analyzed by RT-qPCR. TB Green Premix Ex Taq master mix was used to amplify a portion of Nox4 from cDNA. Amplification curves were obtained using template cDNA equivalent to 10 and 100 ng of RNA (Figure 1), confirming successful amplification of the Nox4 target.

Conclusions

The difficult-to-analyze Nox4 mRNA could be measured by qPCR with TB Green Premix Ex Taq master mix. Previous attempts to analyze Nox4 using a SYBR green master mix were unsuccessful, but amplification using TB Green Premix Ex Taq master mix provided reliable results the first time.

Methods

Total RNA was prepared from THP-1 monocytes using a commercially available RNA extraction kit (Ambion). cDNA was synthesized by reverse transcription with the QuantiTec Reverse Transcription Kit (Qiagen) following the manufacturer's recommended protocol. The cDNA was used for intercalating green dye-based qPCR analysis of a 281-bp portion of Nox4 using TB Green Premix Ex Taq (Tli RNase H Plus) master mix on an ABI 7900HT system (Thermo Fisher Scientific). Reactions were assembled according to the recommended protocol (Table I). Reactions were performed in duplicate. The PCR amplification conditions were 95°C for 30 sec (initial denaturation), then 40 cycles of 95°C for 5 sec, and 60°C for 34 sec.

Component	Amount
TB Green Premix Ex Taq (Tli RNaseH Plus) (2X)	10 µl
Forward Primer (10 µM)	0.4 µl
Reverse Primer (10 µM)	0.4 µl
ROX Reference Dye (50X)	0.4 µl
Template	2.0 µl
Sterile dH₂O	6.8 µl
Total	20 µl

Table I. qPCR reaction composition.

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RR420A

TB Green™ Premix Ex Taq™ (Tli RNase H Plus)

200 Rxns

$327.00

License Statement

ID Number
M40	This product is covered by the claims of U.S. Patent No. 7,422,888 and its foreign counterpart patent claims.
M82	This product is covered by the claims of Japanese patent No. 6029636 and its foreign counterparts.
L11	This product is provided under an intellectual property license from Life Technologies Corporation. The transfer of this product is contingent on the buyer using the purchased product and components of the product solely in research conducted by the buyer (whether the buyer is an academic or for-profit entity). The sale of this product is expressly conditioned on the buyer not using the product or its components for any Commercial Purposes. Commercial Purposes means any activity by a party to generate revenue, which may include, but is not limited to use of the product or its components: (i) in manufacturing; (ii) to provide a service, information, or data in return for payment; (iii) for therapeutic, diagnostic or prophylactic purposes; or (iv) for resale, regardless of whether they are resold for use in research. For information on purchasing a license to this product for purposes other than research, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008 USA or outlicensing@lifetech.com.

TB Green Premix Ex Taq (Tli RNase H Plus) provides reduced PCR inhibition caused by the presence of double-stranded RNA/cDNA hybrids remaining after cDNA synthesis. This inhibition is common when using low or RNase H minus RTs, and with GC-rich templates and/or genes with poor expression. The presence of dsRNA/cDNA hybrids is a frequent cause of poor qPCR amplification and/or reaction failure. TB Green Premix Ex Taq (Tli RNase H Plus) prevents this potential problem at no additional cost and without requiring a separate RNase H digestion step prior to qPCR.

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.

Components

RR420B

TB Green™ Premix Ex Taq™ (Tli RNase H Plus)

400 Rxns

$635.00

License Statement

ID Number
M40	This product is covered by the claims of U.S. Patent No. 7,422,888 and its foreign counterpart patent claims.
M82	This product is covered by the claims of Japanese patent No. 6029636 and its foreign counterparts.
L11	This product is provided under an intellectual property license from Life Technologies Corporation. The transfer of this product is contingent on the buyer using the purchased product and components of the product solely in research conducted by the buyer (whether the buyer is an academic or for-profit entity). The sale of this product is expressly conditioned on the buyer not using the product or its components for any Commercial Purposes. Commercial Purposes means any activity by a party to generate revenue, which may include, but is not limited to use of the product or its components: (i) in manufacturing; (ii) to provide a service, information, or data in return for payment; (iii) for therapeutic, diagnostic or prophylactic purposes; or (iv) for resale, regardless of whether they are resold for use in research. For information on purchasing a license to this product for purposes other than research, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008 USA or outlicensing@lifetech.com.

TB Green Premix Ex Taq (Tli RNase H Plus) provides reduced PCR inhibition caused by the presence of double-stranded RNA/cDNA hybrids remaining after cDNA synthesis. This inhibition is common when using low or RNase H minus RTs, and with GC-rich templates and/or genes with poor expression. The presence of dsRNA/cDNA hybrids is a frequent cause of poor qPCR amplification and/or reaction failure. TB Green Premix Ex Taq (Tli RNase H Plus) prevents this potential problem at no additional cost and without requiring a separate RNase H digestion step prior to qPCR. Cat. # RR420B contains 2 of Cat. # RR420A. Please refer to Cat. # RR420A 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.

RR420L

TB Green™ Premix Ex Taq™ (Tli RNase H Plus), 1 x 5 mL

200 Rxns

$327.00

License Statement

ID Number
M82	This product is covered by the claims of Japanese patent No. 6029636 and its foreign counterparts.
L11	This product is provided under an intellectual property license from Life Technologies Corporation. The transfer of this product is contingent on the buyer using the purchased product and components of the product solely in research conducted by the buyer (whether the buyer is an academic or for-profit entity). The sale of this product is expressly conditioned on the buyer not using the product or its components for any Commercial Purposes. Commercial Purposes means any activity by a party to generate revenue, which may include, but is not limited to use of the product or its components: (i) in manufacturing; (ii) to provide a service, information, or data in return for payment; (iii) for therapeutic, diagnostic or prophylactic purposes; or (iv) for resale, regardless of whether they are resold for use in research. For information on purchasing a license to this product for purposes other than research, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008 USA or outlicensing@lifetech.com.
M40	This product is covered by the claims of U.S. Patent No. 7,422,888 and its foreign counterpart patent claims.

TB Green Premix Ex Taq (Tli RNase H Plus) provides reduced PCR inhibition caused by the presence of double stranded RNA/cDNA hybrids remaining after cDNA synthesis. This inhibition is common when using low or RNase H minus RTs, and with GC rich templates and/or genes with poor expression. The presence of dsRNA/cDNA hybrids is a frequent cause of poor qPCR amplification and/or reaction failure. TB Green Premix Ex Taq (Tli RNase H Plus) prevents this potential problem at no additional cost and without requiring a separate RNase H digestion step prior to qPCR. Cat.# RR420L and RR420W are larger package sizes [5 ml and 25 ml (5 x 5 ml), respectively]. Each provide convenient larger packaging and are supplied with a single, separate vial of ROX Reference Dye.

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.

Components

RR420W

TB Green™ Premix Ex Taq™ (Tli RNase H Plus), 5 x 5 mL

1,000 Rxns

$1299.00

License Statement

ID Number
M82	This product is covered by the claims of Japanese patent No. 6029636 and its foreign counterparts.
L11	This product is provided under an intellectual property license from Life Technologies Corporation. The transfer of this product is contingent on the buyer using the purchased product and components of the product solely in research conducted by the buyer (whether the buyer is an academic or for-profit entity). The sale of this product is expressly conditioned on the buyer not using the product or its components for any Commercial Purposes. Commercial Purposes means any activity by a party to generate revenue, which may include, but is not limited to use of the product or its components: (i) in manufacturing; (ii) to provide a service, information, or data in return for payment; (iii) for therapeutic, diagnostic or prophylactic purposes; or (iv) for resale, regardless of whether they are resold for use in research. For information on purchasing a license to this product for purposes other than research, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008 USA or outlicensing@lifetech.com.
M40	This product is covered by the claims of U.S. Patent No. 7,422,888 and its foreign counterpart patent claims.

TB Green Premix Ex Taq (Tli RNase H Plus) provides reduced PCR inhibition caused by the presence of double-stranded RNA/cDNA hybrids remaining after cDNA synthesis. This inhibition is common when using low or RNase H-minus RTs, and with GC-rich templates and/or genes with poor expression. The presence of dsRNA/cDNA hybrids is a frequent cause of poor qPCR amplification and/or reaction failure. TB Green Premix Ex Taq (Tli RNase H Plus) prevents this potential problem at no additional cost and without requiring a separate RNase H digestion step prior to qPCR. Cat. # RR420W contains five of Cat. # RR420L. Please refer to Cat. # RR420L 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.

Real-time PCR

Tech Note

Data kindly provided by: Hong Seok Kim, Postdoctoral researcher, UT Health Science Center at San Antonio

Introduction

Results

Conclusions

Methods

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