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mRNA and cDNA synthesis

Efficient preparation of cDNA: PrimeScript Reverse Transcriptase

PrimeScript Reverse Transcriptase has exceptionally strong strand-displacement activity and enables efficient preparation of cDNA up to 12 kb in length. It is robust, versatile, and well-suited for applications requiring full-length cDNA, such as preparation of cDNA libraries and other techniques involving first-strand cDNA synthesis (RT-PCR, preparation of cDNA probes, real-time quantitative RT-PCR).

PrimeScript RT can be used to perform reverse transcription from any RNA template, including GC-rich RNAs and those with high levels of secondary structure. This enzyme is a modified, recombinant MMLV (Moloney Murine Leukemia Virus) reverse transcriptase and is verified to be RNase H-minus. Because of the excellent extension capability of PrimeScript RT, preparation of cDNA can be performed at a lower temperature (42°C), decreasing the risk of RNA degradation that can occur during conventional reactions performed at higher temperatures.

First introduced in Japan, PrimeScript is now available to scientists worldwide. Over 3,700 peer-reviewed articles have been published in which PrimeScript Reverse Transcriptase was referenced in a variety of scientific applications such as gene expression profiling, gene discovery, transcriptome analysis, miRNA expression and regulation analysis, molecular evolution studies, virology, and microbiology.

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Cat. #	Product	Size	Price
2680B	PrimeScript™ Reverse Transcriptase	40,000 Units	USD $932.00
PrimeScript Reverse Transcriptase is a modified MMLV (Moloney Murine Leukemia Virus) RTase. This enzyme has extremely high extension capability and can synthesize long first-strand cDNA efficiently. Even for difficult templates, including RNAs with complex secondary structure, it is possible to synthesize first-strand cDNA at the normal reverse transcription temperature (42℃) with this enzyme. It is not necessary to perform the RT reaction at higher temperatures (a condition that may cause RNA degradation). This enzyme is suitable for preparation of long cDNAs, for construction of cDNA libraries that include full-length cDNA, etc. Cat. # 2680B contains 4 of Cat. # 2680A. Please refer to Cat. # 2680A 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 Back 2680B: PrimeScript Reverse Transcriptase Back Strand displacement and elongation activity of PrimeScript Reverse Transcriptase in comparison to Company A RTase Strand displacement and elongation activity of PrimeScript Reverse Transcriptase in comparison to Company A RTase. Top panel: diagram of cDNA synthesis reactions performed using either an oligo dT primer (resulting in a 6.4-kb product) or a gene-specific primer (yielding a 4.4-kb product). Primer extension reactions were performed at 50°C. Bottom panel: Primer extension reactions were analyzed on an alkaline denaturing gel. PrimeScript Reverse Transcriptase provided higher yield of both the 6.4-kb and the 4.4-kb product, demonstrating superior strand displacement capability. Back cDNA synthesis with low background, excellent extension, and high yield at 42°C by PrimeScript Reverse Transcriptase in comparison to six other commercially available RTases cDNA synthesis with low background, excellent extension, and high yield at 42°C by PrimeScript Reverse Transcriptase in comparison to six other commercially available RTases. RNA ladder (1 kb, 2 kb, 4.4 kb, 6.4 kb, 8.4 kb, 10 kb, and 12 kb) was used as template for first-strand cDNA synthesis reactions. Reactions were performed at 42°C according to the recommendations of each manufacturer. Equivalent reaction volumes were analyzed using alkaline denaturing gel electrophoresis and products were detected with green intercalating dye and fluorescence image analysis. Reactions performed using PrimeScript Reverse Transcriptase showed the highest yield of full-length cDNA product and lowest background. Back Highly accurate reverse transcription with PrimeScript Reverse Transcriptase Highly accurate reverse transcription with PrimeScript Reverse Transcriptase. First-strand cDNA synthesis reactions were performed using PrimeScript Reverse Transcriptase or other commercially available RTases. The template for all reactions was Human Placenta Total RNA (Takara Bio). All reactions used an oligo dT primer and were performed according to the recommendations of each manufacturer. After synthesizing cDNA, PCR amplification was performed with highly accurate PrimeSTAR HS DNA Polymerase (target gene: TF, amplification product: 500 bp). A control reaction was included in which PCR was performed (no reverse transcription step). Amplified fragments were cloned and multiple clones from each reaction were sequenced. Error rate was defined as the number of errors per total bases sequenced for each reaction type (~200,000 bases). With only 7 errors out of 201,297 bases (0.0035% error rate), PrimeScript Reverse Transcriptase showed the highest accuracy of all RTases studied.
2680A	PrimeScript™ Reverse Transcriptase	10,000 Units	USD $333.00
PrimeScript Reverse Transcriptase is a modified MMLV (Moloney Murine Leukemia Virus) RTase. This enzyme has extremely high extension capability and can synthesize long first-strand cDNA efficiently. Even for difficult templates, including RNAs with complex secondary structure, it is possible to synthesize first-strand cDNA at the normal reverse transcription temperature (42℃) with this enzyme. It is not necessary to perform the RT reaction at higher temperatures (a condition that may cause RNA degradation). This enzyme is suitable for preparation of long cDNAs, for construction of cDNA libraries that include full-length cDNA, etc. 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 Back 2680A: PrimeScript Reverse Transcriptase Back Strand displacement and elongation activity of PrimeScript Reverse Transcriptase in comparison to Company A RTase Strand displacement and elongation activity of PrimeScript Reverse Transcriptase in comparison to Company A RTase. Top panel: diagram of cDNA synthesis reactions performed using either an oligo dT primer (resulting in a 6.4-kb product) or a gene-specific primer (yielding a 4.4-kb product). Primer extension reactions were performed at 50°C. Bottom panel: Primer extension reactions were analyzed on an alkaline denaturing gel. PrimeScript Reverse Transcriptase provided higher yield of both the 6.4-kb and the 4.4-kb product, demonstrating superior strand displacement capability. Back cDNA synthesis with low background, excellent extension, and high yield at 42°C by PrimeScript Reverse Transcriptase in comparison to six other commercially available RTases cDNA synthesis with low background, excellent extension, and high yield at 42°C by PrimeScript Reverse Transcriptase in comparison to six other commercially available RTases. RNA ladder (1 kb, 2 kb, 4.4 kb, 6.4 kb, 8.4 kb, 10 kb, and 12 kb) was used as template for first-strand cDNA synthesis reactions. Reactions were performed at 42°C according to the recommendations of each manufacturer. Equivalent reaction volumes were analyzed using alkaline denaturing gel electrophoresis and products were detected with green intercalating dye and fluorescence image analysis. Reactions performed using PrimeScript Reverse Transcriptase showed the highest yield of full-length cDNA product and lowest background. Back Highly accurate reverse transcription with PrimeScript Reverse Transcriptase Highly accurate reverse transcription with PrimeScript Reverse Transcriptase. First-strand cDNA synthesis reactions were performed using PrimeScript Reverse Transcriptase or other commercially available RTases. The template for all reactions was Human Placenta Total RNA (Takara Bio). All reactions used an oligo dT primer and were performed according to the recommendations of each manufacturer. After synthesizing cDNA, PCR amplification was performed with highly accurate PrimeSTAR HS DNA Polymerase (target gene: TF, amplification product: 500 bp). A control reaction was included in which PCR was performed (no reverse transcription step). Amplified fragments were cloned and multiple clones from each reaction were sequenced. Error rate was defined as the number of errors per total bases sequenced for each reaction type (~200,000 bases). With only 7 errors out of 201,297 bases (0.0035% error rate), PrimeScript Reverse Transcriptase showed the highest accuracy of all RTases studied.

Overview

Strong strand-displacement and extension capability: capable of synthesizing long and full-length cDNA molecules (up to 12 kb)
Preparation of cDNA at 42°C results in lower background and higher cDNA yields because the lower temperature does not promote RNA degradation
Outstanding accuracy: lowest error rate among five commercially available reverse transcriptases tested
Highly specific: low rates of non-specific annealing, even on incompletely denatured RNA
Works on challenging templates: excellent results even with GC-rich templates and templates with high levels of secondary structure
Highly sensitive: use less of your precious RNA samples
Demonstrated success with reverse transcription reaction times of 30 min (60 min for longer transcripts)

More Information

Applications

RT-PCR
First-strand cDNA synthesis
cDNA probe preparation
Synthesis of cDNA libraries with a high proportion of full-length cDNAs

Standard protocol

Prepare the following mixture in a microtube.

Oligo dT primer* 50 pmol

dNTP Mixture (10 mM each) 1 µl

Template RNA ≤5 µg (total RNA) or ≤1 µg (mRNA)

RNase-free dH₂O to 10 µl

* For Random Primers (6-mers), use 50 pmol. For gene-specific primers, use 2 pmol.
Heat at 65°C for 5 min, then immediately cool on ice.
Prepare the reaction mixture by combining the following components to create a 20-µl reaction.

Template RNA/Primer Mixture 10 µl

5X PrimeScript Buffer 4 µl

RNase Inhibitor 20 U

PrimeScript Reverse Transcriptase 100 U

RNase-free dH₂O to 20 µl
Mix gently.
Perform the reaction under the following conditions.

30°C 10 min*

42°C** 30–60 min

* This step is required for random primers.
** PrimeScript RTase has robust extension capability even with template having complex secondary structure. Therefore, incubation at 42°C is generally recommended. However, for RT-PCR reactions where the reverse primer for PCR is also used as a reverse transcription primer, we recommend performing the reverse transcription reaction at 50°C for 30 min to reduce the possibility of non-specific amplification products.
Heat at 70°C for 15 min and cool on ice.
The resulting product can be used for a second-strand synthesis reaction or as a template for PCR.

Components

Cat. # 2680A:

PrimeScript Reverse Transcriptase	10,000 U
5X PrimeScript Buffer	500 µl

Buffer composition

5X PrimeScript Buffer (for cDNA synthesis)
50 mM	Tris-HCl, pH 8.3
375 mM	KCl
15 mM	MgCl₂

Unit definition

One unit is the amount of the enzyme that incorporates 1 nmol of [³H]dTTP in 10 minutes at 37°C, with poly(rA), oligo(dT) 12–18 as the primer-template.

Reaction mixture for unit definition

50 mM: Tris-HCl, pH 8.3
75 mM: KCl
8 mM: MgCl₂
10 mM: DTT
20 µg/ml: (ra)n (dT)12–18
0.5 mM: [³H]dTTP
0.1%: NP-40

Storage buffer composition

20 mM Tris-HCl, pH 7.8
100 mM NaCl
1 mM EDTA
1 mM DTT
50% Glycerol (v/v)

Choosing a PrimeScript kit for endpoint RT-PCR

If you want to...	Use this...
Prepare full-length cDNA using your own primers	PrimeScript Reverse Transcriptase (Cat. # 2680A and 2680B)
Prepare full-length cDNA using a kit that includes primers	PrimeScript First Strand cDNA Synthesis Kit (Cat. # 6110A and 6110B)
Perform 2-step RT-PCR with a kit that includes RT, primers, and PCR enzyme	PrimeScript RT-PCR Kit (Cat. # RR014A and RR014B)
Perform cDNA cloning studies with extremely high fidelity	PrimeScript High Fidelity RT-PCR Kit (Cat. # R022A and R022B)
Perform 1-step PCR	PrimeScript One-Step RT-PCR Kit, Ver. 2 (Cat. # RR055A and RR055B)

Additional product information

Please see the product's Certificate of Analysis for information about storage conditions, product components, and technical specifications. Please see the Kit Components List to determine kit components. Certificates of Analysis and Kit Components Lists are located under the Documents tab.

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FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. © 2025 Takara Bio Inc. All Rights Reserved. All trademarks are the property of Takara Bio Inc. or its affiliate(s) in the U.S. and/or other countries or their respective owners. Certain trademarks may not be registered in all jurisdictions. Additional product, intellectual property, and restricted use information is available at takarabio.com.

Oligo dT primer*	50 pmol
dNTP Mixture (10 mM each)	1 µl
Template RNA	≤5 µg (total RNA) or ≤1 µg (mRNA)
RNase-free dH₂O	to 10 µl

Template RNA/Primer Mixture	10 µl
5X PrimeScript Buffer	4 µl
RNase Inhibitor	20 U
PrimeScript Reverse Transcriptase	100 U
RNase-free dH₂O	to 20 µl

30°C	10 min*
42°C**	30–60 min